Deglycosylation methods for electrophoresis of glycosylated proteins

ABSTRACT

The disclosure relates to methods of analyzing a post-translationally modified protein of interest using electrophoresis, the methods comprising deglycosylating the protein of interest after labeling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisionalpatent Application Ser. No. 62/963,646 filed on Jan. 21, 2020, thecontents of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The disclosure relates to the fields of biochemistry, molecular biologyand the analysis of proteins via electrophoresis.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:REGE-019-001US_SeqList_ST25.txt, date recorded: Jan. 19, 2021, file size1 kilobyte).

BACKGROUND

Capillary based electrophoresis (CE) and microchip based capillaryelectrophoresis (MCE) are common analytical methods in thepharmaceutical industry used to characterize therapeutic proteinintegrity and purity based on protein size, and provide quality control.While standard, industry recommended sample preparation methods workwell for many proteins, heavily glycosylated proteins are problematicdue to poor separation and quantification by CE and MCE. In addition,partially glycosylated peaks and non-glycosylated peak in the MCEprofile may overlap with impurity peaks and interfere withquantification. There thus exists a need in the art for additionalsample preparation methods that can overcome the challenges of workingwith glycosylated proteins. This invention provides methods for labelingheavily glycosylated proteins that can be used to prepare proteins foranalysis by electrophoresis methods such as CE and MCE.

SUMMARY

The disclosure provides methods of analyzing a sample comprising aprotein of interest, the methods comprising denaturing, fluorescentlylabeling, quenching and deglycosylating the sample; wherein thedenaturing, labeling and quenching steps occur prior to deglycosylation.The methods of analyzing a sample of the disclosure can reduce oreliminate electropherogram peaks due to endoglycosidase, and can reducefree dye interference, thereby providing fast, accurate and highlyreproducible and high throughput methods through which glycoproteins canbe analyzed.

The disclosure provides methods of analyzing a sample comprising aprotein of interest, the methods comprising: (a) denaturing the sample;(b) labeling the sample with a fluorescent label to produce a labeledsample; (c) quenching un-reacted fluorescent label in the labeledsample; (d) deglycosylating the labeled sample with an endoglycosidase;and (e) performing electrophoresis on the labeled sample; wherein thesample is denatured, labeled and quenched in steps (a) through (c) priorto deglycosylation in step (d).

In some embodiments of the methods of the disclosure, the protein ofinterest comprises at least one glycosylation site. In some embodiments,the protein is of interest is a glycosylated protein. In someembodiments, the glycosylated protein comprises at least one attachedglycan. In some embodiments, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5% or at least 10% of the total weight of theglycosylated protein comprises glycans (10% w/w).

In some embodiments of the methods of the disclosure, the protein ofinterest comprises an antigen binding domain. In some embodiments, theprotein of interest comprises an antibody, an antibody fragment or anscFv. In some embodiments, the protein of interest comprises an Fcdomain. In some embodiments, the protein of interest comprises areceptor fusion protein. In some embodiments, the receptor fusionprotein is a receptor-Fc-fusion protein or a soluble TCR-Fc fusionprotein. In some embodiments, the receptor fusion protein is a trapprotein or a mini trap protein. In some embodiments, the protein ofinterest is a trap protein or a mini trap protein. In some embodiments,the protein of interest is a recombinant human protein.

In some embodiments of the methods of the disclosure, the glycosylationsite comprises an Asn-X-Ser/Thr consensus sequence. In some embodiments,the at least one attached glycan is N-linked. In some embodiments, theat least one attached glycan is N-linked to an asparagine in theglycosylated protein. In some embodiments, the endoglycosidase catalyzesdeglycosylation of N-linked glycans. In some embodiments, theendoglycosidase is selected from the group consisting ofPeptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H),Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1,Endoglycosidase F2 and Endoglycosidase F4. In some embodiments, theendoglycosidase is PNGase F. In some embodiments, the PNGase F is RapidPNGase F. In some embodiments, the Rapid PNGase F is non-reducing. Insome embodiments, the PNGase F is reducing.

In some embodiments of the methods of the disclosure, deglycosylatingthe sample comprises heating the sample to about 35° C. for 30 minutes.In some embodiments, deglycosylating the sample comprises heating thesample to about 50° C. for between 10 and 30 minutes. In someembodiments, deglycosylating the sample comprises heating the sample toabout 50° C. for 10 minutes. In some embodiments, deglycosylating thesample comprises a reaction mixture comprising between 0.2-1.5 mglabeled protein of interest, and between 1-5 μL Rapid PNGase F in a 10μL reaction volume, excluding the volume of the Rapid PNGase F. In someembodiments, the reaction mixture comprises 0.2 mg labeled protein ofinterest. In some embodiments, the reaction mixture comprises 5 μL RapidPNGase F. In some embodiments, the reaction mixture comprises a buffer.

In some embodiments of the methods of the disclosure, the at least oneglycan is an O-linked glycan. In some embodiments, the endoglycosidasecatalyzes deglycosylation of O-linked glycans. In some embodiments, theendoglycosidase comprises Endo-α-N-acetylgalactosamindase(O-glycosidase).

In some embodiments of the methods of the disclosure, labeling thesample with the fluorescent label comprises heating the sample to about35° C. for 10-30 minutes. In some embodiments, labeling the sample withthe fluorescent label comprises heating the sample to about 35° C. for15 minutes.

In some embodiments of the methods of the disclosure, the sample isdenatured using a reducing solution. In some embodiments, the reducingsolution comprises dithiothreitol (DTT). In some embodiments, the sampleis denatured using a non-reducing solution. In some embodiments, thenon-reducing solution comprises iodoacetamide (IAM). In someembodiments, denaturing the sample comprises heating the sample tobetween 40° C. and 99° C. for between 1 minute and 5 hours. In someembodiments, denaturing the sample comprises heating the sample tobetween 50° C. and 99° C. for between 1 to 60 minutes.

In some embodiments of the methods of the disclosure, quenching theun-reacted fluorescent label comprises adding a stop solution.

In some embodiments of the methods of the disclosure, the methodsfurther comprise analyzing a reference standard in parallel to thesample.

In some embodiments of the methods of the disclosure, theelectrophoresis is selected from the group consisting of gelelectrophoresis, isoelectric focusing, capillary electrophoresis (CE) ormicrochip capillary electrophoresis (MCE). In some embodiments, theelectrophoresis is MCE. In some embodiments, the MCE is carried outusing an MCE instrument.

In some embodiments of the methods of the disclosure, methods result inreduced free dye interference in the less than 20 kDa range and areduced or absent endoglycosidase peak in an electropherogram whencompared to an electropherogram generated using a sample labeled afterdeglycosylation. In some embodiments, the endoglycosidase peak isreduced by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% when compared to an electropherogram generated using a samplelabeled after deglycosylation. In some embodiments, the endoglycosidasepeak is absent in an electropherogram when compared to anelectropherogram generated using a sample labeled after deglycosylation.

The disclosure provides methods of determining stability of a protein ofinterest comprising: (a) stressing a sample comprising the protein ofinterest; (b) denaturing the stressed sample and a non-stressed samplecomprising the protein of interest; (c) labeling the stressed sample andthe non-stressed sample with a fluorescent label to produce a labeledstressed sample and a labeled non-stressed sample; (d) quenchingun-reacted fluorescent label in the labeled stressed sample and thelabeled non-stressed sample; (e) deglycosylating the labeled stressedsample and the labeled non-stressed sample with an endoglycosidase; (f)performing microchip capillary electrophoresis (MCE) on the labeledstressed sample and the labeled non-stressed sample to generateelectropherograms for the stressed sample and the non-stressed sample;and (g) comparing the electropherograms from the stressed sample and thenonstressed sample, thereby determining the stability of the protein ofinterest; wherein the stressed sample and the non-stressed sample aredenatured, labeled and quenched in steps (b) through (d) prior todeglycoslation in step (e).

In some embodiments of the methods of the disclosure, stressing thesample comprises thermally stressing the sample. In some embodiments,thermally stressing the sample comprises holding the sample at betweenabout 30° C. and about 45° C. for at least 1 week, at least 2 weeks, atleast 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, atleast 7 weeks or at least 8 weeks.

In some embodiments of the methods of the disclosure, stressing thesample comprises at least one freeze/thaw cycle.

In some embodiments of the methods of the disclosure, stressing thesample comprises exposing the sample to storage conditions. In someembodiments, the storage conditions comprise a temperature of about −80°C. to −30° C. for at least 1 week, at least 2 weeks, at least 3 weeks,at least 1 month, at least 2 months, at least 3 months, at least 6months, at least 8 months, at least 12 months, at least 18 months, atleast 24 months or at least 30 months. In some embodiments, the storageconditions comprise a temperature of about 2° C. to 8° C. for at least 1week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2months, at least 3 months, at least 6 months, at least 8 months, atleast 12 months or at least 18 months.

In some embodiments of the methods of the disclosure, stressing thesample comprises mechanically agitating the sample.

In some embodiments of the methods of the disclosure, stressing thesample comprises lyophilizing and rehydrating the sample.

In some embodiments of the methods of the disclosure, stressing thesample comprises exposing the sample to light, radiation, singlet oxygenspecies, free radicals, high pH conditions or low pH conditions.

In some embodiments of the methods of the disclosure, the protein ofinterest comprises at least one glycosylation site. In some embodiments,the protein is of interest is a glycosylated protein. In someembodiments, the glycosylated protein comprises at least one attachedglycan. In some embodiments, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5% or at least 10% of the total weight of theglycosylated protein comprises glycans (10% w/w). In some embodiments,at least 10% of the total weight of the glycosylated protein comprisesglycans (10% w/w).

In some embodiments of the methods of the disclosure, the protein ofinterest comprises an antigen binding domain. In some embodiments, theprotein of interest comprises an antibody, an antibody fragment or anscFv. In some embodiments, the protein of interest comprises an Fcdomain. In some embodiments, the protein of interest comprises areceptor fusion protein. In some embodiments, the receptor fusionprotein is a receptor-Fc-fusion protein or a soluble TCR-Fc fusionprotein. In some embodiments, the receptor fusion protein is a trapprotein or a mini trap protein. In some embodiments, the protein ofinterest is a trap protein or a mini trap protein. In some embodiments,the protein of interest is a recombinant human protein.

In some embodiments of the methods of the disclosure, the glycosylationsite comprises an Asn-X-Ser/Thr consensus sequence. In some embodiments,the at least one attached glycan is N-linked. In some embodiments, theat least one attached glycan is N-linked to an asparagine in theglycosylated protein. In some embodiments, the endoglycosidase catalyzesdeglycosylation of N-linked glycans. In some embodiments, theendoglycosidase is selected from the group consisting ofPeptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H),Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1,Endoglycosidase F2 and Endoglycosidase F4. In some embodiments, theendoglycosidase is PNGase F. In some embodiments, the PNGase F is RapidPNGase F. In some embodiments, the Rapid PNGase F is non-reducing. Insome embodiments, the Rapid PNGase F is reducing.

In some embodiments of the methods of the disclosure, deglycosylatingthe stressed and non-stressed samples comprises heating the samples toabout 35° C. for 30 minutes. In some embodiments, deglycosylating thestressed and non-stressed samples comprises heating the samples to about50° C. for between 10 and 30 minutes. In some embodiments,deglycosylating the stressed and non-stressed samples comprises heatingthe samples to about 50° C. for 10 minutes. In some embodiments,deglycosylating the stressed and non-stressed samples comprises areaction mixture for each sample comprising between 0.2-1.5 mg labeledprotein of interest, and between 1-5 μL Rapid PNGase F in a 10 μLreaction volume excluding the volume of the Rapid PNGase F. In someembodiments, the reaction mixture for each of the stressed andnon-stressed samples comprises 5 μL Rapid PNGase F. In some embodiments,each of the stressed and non-stressed sample comprise 0.2 mg labeledprotein of interest. In some embodiments, the reaction mixture for eachof the stressed and non-stressed samples comprises a buffer.

In some embodiments of the methods of the disclosure, the at least oneglycan is an O-linked glycan. In some embodiments, the endoglycosidasecatalyzes deglycosylation of O-linked glycans. In some embodiments, theendoglycosidase comprises Endo-α-N-acetylgalactosamindase(O-glycosidase).

In some embodiments of the methods of the disclosure, labeling thestressed and non-stressed samples with the fluorescent label comprisesheating each sample to about 35° C. for 30 minutes.

In some embodiments of the methods of the disclosure, the stressed andnon-stressed samples are denatured using a reducing solution. In someembodiments, the reducing solution comprises dithiothreitol (DTT). Insome embodiments, the stressed and non-stressed samples are denaturedusing a non-reducing solution. In some embodiments, the non-reducingsolution comprises iodoacetamide (IAM). In some embodiments, denaturingthe stressed and non-stressed samples comprises heating the samples tobetween 40° C. and 99° C. for between 1 minute and 5 hours. In someembodiments, denaturing the stressed and non-stressed samples comprisesheating the samples to between 50° C. and 99° C. for between 1 to 60minutes.

In some embodiments of the methods of the disclosure, quenching theun-reacted fluorescent label comprises adding a stop solution.

In some embodiments of the methods of the disclosure, the methodsfurther comprise analyzing a reference standard in parallel to thestressed and non-stressed samples. In some embodiments, comparing theelectropherograms for the stressed and non-stressed samples comprisescomparing peak number, height, position, area, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a diagram showing protocols for Method A, withoutdeglycosylation; Method B, deglycosylation prior to labeling; and MethodC, deglycosylation after labeling. NR: non-reducing, R: reducing, MC:microchip capillary electrophoresis.

FIG. 2 is an electropherogram generated using Protein 1 undernon-reduced conditions, using a protocol without deglycosylation (MethodA, shown in red), and a protocol with deglycosylation prior proteinlabeling (Method B, shown in blue). The numeric peak labels indicate themolecular weight of the proteins as measured by microchip capillaryelectrophoresis (MCE). The Rapid PNGase F (PNGase) peak appears in theelectropherogram where indicated.

FIG. 3 shows three electropherograms generated using Protein 1 undernon-reduced conditions using Method B (deglycosylation prior tolabeling). Protein 1 samples were treated with thermal stress prior toanalysis at 37° C. for no time (0, red, top), 2 weeks (blue, middle), or4 weeks (black, bottom). The low molecular weight 1 peak (LMW 1) peakincreased with stress, and merged with PNGase peak.

FIG. 4 is an electropherogram generated using Protein 1 under reducingconditions, using a protocol without deglycosylation (Method A, shown inred), and a protocol with deglycosylation before protein labeling(Method B, shown in blue). The numeric peak labels indicate themolecular weight of the proteins as measured by MCE. The PNGase F peakappears in the method B electropherogram, as indicated. The gray shadedbox indicates free dye peaks.

FIG. 5 is a pair of electropherograms generated with Protein 1, in whichProtein 1 was deglycosylated after labeling (Method C). Top: thedeglycosylation reaction was carried out with 1 μL Rapid™ PNGase, at 50°C., for 10, 15, 20 and 30 minutes. Bottom: the deglycosylation reactionwas carried out with 2 μL Rapid™ PNGase, at 50° C., for 10, 15, 20 and30 minutes.

FIG. 6 is an electropherogram generated using Method C and Protein 1,showing the results of deglycosylation with 1, 2, 3, or 4 μL of Rapid™PNGase F in a reaction held at 50° C., for 10 minutes. Inset shows theProtein 1 incompletely deglycosylated peak (right hand shoulder to themain peak), with the arrow indicating a reduction in glycosylatedprotein with increased amounts of Rapid™ PNGase. Free dye peaks areindicated by the gray shaded box. MP, main peak; LMW 1, low molecularweight 1 peak.

FIG. 7 is a series of four electropherograms generated using Method C(deglycosylation after labeling) and Protein 1, which was deglycosylatedwith 1, 2, 3, or 4 of Rapid™ PNGase (from top to bottom). Low molecularweight (LMW) peaks 1-5, main peak (MP), and high molecular weight peak(HMW) are indicated.

FIG. 8 is an electropherogram generated from Method C (deglycosylationafter labeling) using Protein 1 that was thermally stressed by holdingthe protein at 37° C. for 4 weeks (37C 4 w, Black) and non-stressedProtein 1 (t=0, Red). Deglycosylation was carried out using Rapid™PNGase F.

FIG. 9 is an electropherogram generated using Protein 2, which comparesProtein 2 labeled with deglycosylation (Method C) and withoutdeglycosylation (Method A), under non-reduced conditions. PNGase F hasan expected size of 37 KDa, and this peak is not present. Free dye peaksare indicated by the shaded box.

FIG. 10 is an electropherogram generated using Protein 3, which comparesProtein 3 treated with deglycosylation after labeling (blue, Method C)and without deglycosylation treatment (red, Method A), under non-reducedconditions. The numeric peak labels indicate the molecular weight ofproteins and protein fragments measured by MCE.

FIG. 11 is an electropherogram comparing Protein 3, which was treatedwith deglycosylation after labeling (blue, Method C) and withoutdeglycosylation (red, Method A), under reduced conditions. The numericpeak labels indicate the molecular weight of proteins and proteinfragments measured by MCE.

FIG. 12 is an electropherogram comparing Protein 4 withoutdeglycosylation (Method A, red) and deglycosylated after labeling(Method C, blue). Protein 4 was denatured using non-reducing (NR)conditions. The numeric peak labels indicate the molecular weightmeasured by MCE. LMW: low molecular weight; DGMP: Deglycosylated MainPeak; GMP: Glycosylated Main Peak. Free dye peaks are indicated by theshaded box.

FIG. 13 is an electropherogram comparing Protein 4 labeled withoutdeglycosylation (Method A, red) and deglycosylated after labeling(Method C, blue). Protein 4 was denatured using reducing (R) conditions.LC: Light Chain; DHC: Deglycosylated Heavy Chain; GHC: GlycosylatedHeavy Chain. Free dye peaks are indicated by the shaded box.

FIG. 14 shows three electropherograms assaying the effect ofphoto-stress on protein stability that were generated under non-reducedconditions, using Method C and Protein 1. Protein 1 was photo-stressedunder cool white (CW) fluorescent lamp light with 1.2 million lux hours(MLH) accumulative exposure (blue, middle), and 2.4 MLH accumulativeexposure (black, bottom), and compared to non-stressed Protein 1 (red,top). Deglycosylation was carried out using Rapid™ PNGase F. LMW: lowmolecular weight; MP: main peak; HMW: high molecular weight.

FIG. 15 shows three electropherograms assaying the effect ofphoto-stress on protein stability that were generated under non-reducedconditions, using Method C and Protein 1. Protein 1 was photo-stressedunder integrated near ultraviolet (UVA) energy of 200 watt hours/squaremeter (blue, middle), and 400 watt hours/square meter (black, bottom),and compared to non-stressed Protein 1 (red, top). Deglycosylation wascarried out using Rapid™ PNGase F.LMW: low molecular weight; MP: mainpeak; HMW: high molecular weight.

DETAILED DESCRIPTION

The present disclosure provides new methods for preparing a samplecomprising a protein of interest for analysis via electrophoresis. Inthe methods provided herein, the protein of interest is denatured,followed by covalent labeling of the protein using a fluorescent dye,and subsequently quenching the labeling reaction. Following labeling,the labeled protein is contacted with an enzyme such as anendoglycosidase to remove glycans from the protein of interest withoutfurther purification. Unlike previous methods of preparing glycosylatedproteins for electrophoresis, which deglycosylate the proteins prior tolabeling, the methods described herein allow for clear separation ofprotein and peptide species based on mass. These methods also eliminateinterference from the enzyme used in deglycosylation, and free dye fromthe labeling reaction, in microchip electrophoresis (MCE)electropherograms. The methods are fast, highly reproducible and highthroughput, and have been successfully used to analyze glycosylatedproteins. Without wishing to be bound by theory, it is thought that themethods described herein are advantageous with respect to heavilyglycosylated proteins, as heavy glycosylation interferes with migrationof the protein in the MCE or capillary electrophoresis (CE) analysisplatforms, resulting in incorrect measurements of protein molecularweight and imprecise electropherogram peaks. The methods describe hereincan be used in a platform approach which is applicable to anyglycosylated proteins analyzed by methods such as CE and MCE, and tocharacterize the proteins or for quality control purposes. For example,the methods described herein can be used to measure the stability of aprotein of interest when subjected to various conditions, such prolongedholding times at various temperatures, or different formulations.

Accordingly, the disclosure provides methods of preparing a samplecomprising a protein of interest for analysis using electrophoresis,comprising (a) denaturing the sample; (b) labeling the sample with afluorescent label to produce a labeled sample; (c) quenching un-reactedfluorescent label in the labeled sample; (d) deglycosylating the labeledsample with an endoglycosidase; and (e) performing electrophoresis onthe labeled sample; wherein the sample is labeled and quenched in steps(b) and (c) prior to deglycosylation in step (d). In some embodiments,the electrophoresis is microchip capillary electrophoresis (MCE), andthe output is an electropherogram.

Definitions

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the presently claimed invention (especially in thecontext of the claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

Use of the term “about” is intended to describe values either above orbelow the stated value in a range of approx. +/−10%; in otherembodiments the values may range in value either above or below thestated value in a range of approx. +/−5%; in other embodiments thevalues may range in value either above or below the stated value in arange of approx. +/−2%; in other embodiments the values may range invalue either above or below the stated value in a range of approx.+/−1%. The preceding ranges are intended to be made clear by context,and no further limitation is implied.

As used herein, “protein” refers to a molecule comprising two or moreamino acid residues joined to each other by a peptide bond. Proteinsinclude polypeptides and peptides, and may also include modificationssuch as glycosylation, lipid attachment, sulfation, gamma-carboxylationof glutamic acid residues, alkylation, hydroxylation andADP-ribosylation. Proteins can be of scientific or commercial interest,including protein-based drugs, and proteins include, among other things,enzymes, ligands, receptors, antibodies and chimeric or fusion proteins.Proteins are produced by various types of recombinant cells usingwell-known cell culture methods, and are generally introduced into thecell by genetic engineering techniques (e.g., such as a sequenceencoding a chimeric protein, or a codon-optimized sequence, anintronless sequence, etc.) where it may reside as an episome or beintegrated into the genome of the cell.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Denaturing

The disclosure provides methods of denaturing a protein of interest in asample. Denaturing proteins involves the disruption of secondary andtertiary protein structures under conditions insufficient to disruptpeptide bonds, leaving the primary structure intact.

Methods of denaturing a protein of interest under both reducing andnon-reducing conditions are within the scope of the disclosure.

A protein reducing agent is an agent that disrupts disulfide bonds.These disulfide bonds can be within a single polypeptide, or betweenmultiple subunits of a protein encoded on separate polypeptide.Disrupting disulfide bonds between subunits allows for the analysis ofthe individual subunits of a multi-subunit protein to be analyzedindividually. Reducing agents will be known to persons of ordinary skillin the art. Exemplary reducing agents include. but are not limited to,dithiothreitol (DTT, CAS 3483-12-3), beta-mercaptoethanol (BME, 2BME,2-ME, b-mer, CAS 60-24-2), 2-aminoethanethiol (2-MEA-HCl, also calledcysteamine-HCl, CAS 156-57-0), Tris (2-carboxyethyl) phosphinehydrochloride, (TCEP, CAS 5961-85-3), cysteine hydrochloride (Cys-HCl,CAS 52-89-1), or 2-mercaptoethanesulfonic acid sodium salt (MESNA).Other methods for reducing protein bonds are known in the art, such asan immobilized reductant column which contains resin to which athiol-based reducing agent has been immobilized to enable thesolid-phase reduction of peptide and protein disulfide bonds. Reducingagents, including oxidizing agents, are suitable for reducing chemicalinteraction between polypeptides are also envisioned.

In some embodiments, the protein of interest is denatured using areducing solution. In some embodiments, the reducing solution contains135 to 155 mM dithiothreitol (DTT). In some embodiments, the reducingsolution further comprises sodium phosphate and lithium dodecyl sulfate.In some embodiments, the reducing solution comprises or consistsessentially of 0.69% lithium dodecyl sulfate (LDS), 69 mM sodiumphosphate, and 142 mM dithiothreitol. In some embodiments, the reducingsolution contains 40-120 mM DTT, 40-80 mM sodium phosphate and 0.5% to2.0% LDS. In some embodiments, the reducing solution contains 60-100 mMDTT, 50-70 mM sodium phosphate and 0.75% to 1.5% LDS. In someembodiments, the reducing solution contains about 80 mM DTT, about 60 mMsodium phosphate and about 1.2% LDS. In some embodiments, the reducingsolution is added to the sample comprising the protein of interest at aratio of about 1:4 by volume.

In some embodiments, the protein of interest is denatured using anon-reducing solution, i.e. under conditions which preserve disulfidebonds in the protein of interest. In some embodiments, the non-reducingsolution comprises iodoacetamide (IAM). In some embodiments, thenon-reducing solution comprises between 100 and 200 mM iodoacetamide. Insome embodiments, the non-reducing solution further comprises sodiumphosphate and lithium dodecyl sulfate (LDS). In some embodiments, thenon-reducing solution comprises 166 mM iodoacetamide (IAM). In someembodiments, the non-reducing solution comprises, or consistsessentially of, 166 mM iodoacetamide, 0.81% lithium dodecyl sulfate and81 mM sodium phosphate. In some embodiments, the non-reducing solutioncomprises 100-300 mM iodoacetamide, 40-80 mM sodium phosphate, and 0.5%to 2.0% LDS. In some embodiments, the non-reducing solution comprises,150-250 mM iodoacetamide, 50-70 mM sodium phosphate, and 0.75% to 1.5%LDS. In some embodiments, the non-reducing solution comprises about 200mM iodoacetamide, about 60 mM sodium phosphate, and about 1.2% LDS. Insome embodiments, the non-reducing solution is added to the samplecomprising the protein of interest at a ratio of about 1:4 by volume.

In some embodiments, denaturing the sample comprises adding a reducingor non-reducing solution to the sample, and heating the combined sampleand reducing or non-reducing solution. In some embodiments, the sampleis denatured by heat. For example, the combined sample and reducing ornon-reducing solution can be heated to between 30° C. and 99° C.,between 30° C. and 90° C., between 30° C. and 80° C., between 30° C. and70° C., between 30° C. and 60° C., between 30° C. and 50° C., between30° C. and 40° C., between 40° C. and 99° C., between 40° C. and 90° C.,between 40° C. and 80° C., between 40° C. and 70° C., between 40° C. and60° C., between 40° C. and 50° C., between 50° C. and 99° C., between50° C. and 90° C., between 50° C. and 80° C., between 50° C. and 70° C.,or between 50° C. and 60° C. In some embodiments, the combined sampleand reducing or non-reducing solution can be heated for between 1 minuteand 12 hours, between 1 minute and 10 hours, between 1 minute and 5hours, between 1 minute and 4 hours, between 1 minute and 3 hours,between 1 minute and 2 hours, between 1 minute and 60 minutes, between 1minute and 30 minutes, between 1 minute and 15 minutes, between 1 minuteand 10 minutes, between 1 minute and 5 minutes, between 5 minute and 60minutes, between 5 minutes and 30 minutes, between 5 minute and 15minutes, between 5 minute and 10 minutes, between 10 minute and 60minutes, between 10 and 45 minutes, between 10 minutes and 30 minutes,or between 10 minutes and 15 minutes. In some embodiments, the combinedsample and reducing or non-reducing solution can be heated to between40° C. and 99° C. for between 1 minute and 60 minutes. In someembodiments, the combined sample and reducing or non-reducing solutioncan be heated to between 50° C. and 99° C. for between 1 minute and 60minutes. As a further example, the combined sample and reducing ornon-reducing solution can be heated to between 60° C. and 85° C. forbetween 5 to 30 minutes. Alternatively, the combined sample and reducingor non-reducing solution can be heated to 75° C. for 10 minutes. In someembodiments, the combined sample and reducing or non-reducing solutionis heated to 70° C. for 10 minutes.

Deglycosylation

The disclosure provides methods of deglycosylating a protein of interestin a sample. In some embodiments, the protein of interest isdeglycosylated after being labeled with a fluorescent label using themethods described herein. Deglycosylation can be performed using anenzyme such as an endoglycosidase.

Glycoproteins are proteins which contain oligosaccharide chains(glycans) covalently attached to amino acid side-chains. Theseoligosaccharide chains are attached to the protein in a cotranslationalor posttranslational modification.

As used herein, the term “glycan” sometimes used interchangeably with“polysaccharide” and “oligosaccharide” refers to a compound comprisingor consisting of glycosidically linked monosaccharides. The term glycancan also be used to refer to a carbohydrate linked to a glycoprotein orglycolipid, even if the carbohydrate is a monosaccharide. Glycans maycomprise O-glycosidic linkages of monosaccharides. Glycans can be homo-or heteropolymers of monosaccharides, and can be linear or branched.Exemplary glycans can comprise monomers of mannose, N-Acetylglucosamine(GlcNAc), N-Glycolylneuraminic acid (Neu5Gc), galactose, sialic acid,and fucose, among others.

Glycans can be linked to a protein of interest via either N-linkages orO-linkages, and a protein of interest can comprise N-linked glycans,O-linked glycans or a combination of N-linked and O-linked glycans. Asreferred to herein, “N-linked glycans” or “N-linked glycosylation”refers to the attachment of a sugar monomer or polysaccharide to anitrogen atom such as the amide nitrogen of an asparagine (Asn) aminoacid of a protein. As used herein, “O-linked glycans” or “O-linkedglycosylation” refers to the attachment of a sugar monomer orpolysaccharide to the oxygen atom of a serine (Ser) or threonine (Thr)amino acid of a protein. Exemplary O-linked glycans include, but are notlimited to, O—N-acetylgalactosamine (O-GalNAc), O—N-acetylglucosamine(O-GlcNAc), O-Mannose, O-Galactose, O-Fucose and O-Glucose.

Endoglycosidases are enzymes that that hydrolyze internal glycosidicbonds in oligosaccharides. When the oligosaccharides are part of aglycoprotein, the oligosaccharides are released from the glycoproteinthereby.

As used herein, an “endoglycosidase” refers to an enzyme that releasesglycans from glycoproteins or glycolipids. Endoglycosidases may cleavepolysaccharide changes between residues that are not the terminalresidue, and are thus capable of releasing long chain carbohydrates fromtheir cognate protein conjugates. Exemplary endoglycosidases include,but are not limited to, Peptide-N-Glycosidase F (PNGase F),Endoglycosidase H (Endo H), Endoglycosidase S (Endo S). EndoglycosidaseD, Endoglycosidease F1, Endoglycosidase F2, Endoglycosidase F3,O-glycosidase and Endo-β-Galactosidase.

In some embodiments, the endoglycosidase catalyzes the deglycosylationof N-linked glycans. Exemplary endoglycosidases that target N-linkedglycans include, but are not limited to, Peptide-N-Glycosidase F (PNGaseF), Endoglycosidase H (Endo H), Endoglycosidase S (Endo S),Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2 andEndoglycosidase F4. In some embodiments, for example those embodimentswherein the protein of interest comprises N-linked glycans, theendoglycosidase is PNGase F.

In some embodiments, the endoglycosidase catalyzes the deglycosylationof O-linked glycans. Exemplary endoglycosidases that target O-linkedglycans include, but are not limited to,Endo-α-N-Acetylgalactosaminidase (O-glycosidase).

In some embodiments, the endoglycosidase is PNGAse F. PNGase F is anamidase that cleaves between the innermost N-Acetyl-D-Glucosamine(GlcNAc) and asparagine residues of high mannose, hybrid and complexoligosaccharides in N-linked glycoproteins. In some embodiments, thePNGase F is recombinant. In some embodiments, the PNGase F is Rapid™PNGAse F. Rapid™ PNGase F is known in the art and is available from NewEngland Biolabs and other vendors. In some embodiments, the Rapid™PNGase F is in a non-reducing format that preserves disulfide bonds inthe protein of interest. In some embodiments, the Rapid™ PNGase F is ina reducing format that does not preserve disulfide bonds in the proteinof interest.

In some embodiments, deglycosylating the sample comprises a reactionmixture comprising between 0.1 and 3.0 mg labeled protein of interest.In some embodiments, the reaction mixture comprises between 0.1 and 2.0mg labeled protein of interest. In some embodiments, the reactionmixture comprises between 0.1 and 1.5 mg protein of interest. In someembodiments, the reaction mixture comprises between 0.5 and 1.5 mgprotein of interest. In some embodiments, the reaction mixture comprises0.2 mg labeled protein of interest. In some embodiments, the reactionmixture comprises between 1 and 7 μL of Rapid™ PNGase F enzyme in a 10μL reaction volume, excluding the volume of the enzyme. In someembodiments, the reaction mixture comprises between 1 and 5 μL of Rapid™PNGase F enzyme in a 10 μL reaction volume, excluding the volume of theenzyme. In some embodiments, the reaction mixture comprises 1 μL, 2 μL,3 μL, 4 μL, 5 μL, 6 μL or 7 μL of Rapid™ PNGase F enzyme added to a 10μL volume comprising the labeled protein of interest. In someembodiments, the reaction mixture comprises 5 μL of Rapid™ PNGase Fenzyme in a 10 μL reaction volume, excluding the volume of the enzyme.In some embodiments, the reaction mixture comprises 5 μL Rapid™ PNGase Fenzyme added to a 10 μL volume comprising the labeled protein ofinterest. In some embodiments, the reaction mixture comprises anadditional buffer, for example a reaction buffer that facilitates theaction of the PNGase F enzyme. In some embodiments, the reaction mixturedoes not comprise an additional buffer.

In some embodiments, for example those embodiments where theendoglycosidase is PNGase F, deglycosylating the sample comprisesheating the sample to 25° C. to 65° C. for between 100 and 60 minutes.In some embodiments, deglycosylating the sample comprises heating thesample to 30° C. to 50° C. for between 20 and 40 minutes. In someembodiments, deglycosylating the sample comprises heating the sample to35° C. for 30 minutes.

In some embodiments, for example those embodiments where theendoglycosidase is Rapid™ PNGase F, deglycosylating the sample comprisesheating the sample 50° C. for between 10 and 30 minutes. In someembodiments, deglycosylating the sample comprises heating the sample 50°C. for 10 minutes.

Protein Labeling

The disclosure provides methods of labeling proteins of interest. Insome embodiments, the proteins of interest are labeled prior todeglycosylation. In some embodiments, proteins of interest are labeledwith a fluorescent label, such as a fluorescent dye. Any suitable labelis envisaged as within the scope of the disclosure.

As used herein, “detectable label” or “label” refers to a chemical usedto facilitate identification and/or quantitation of a target substance,such as a protein of interest. Illustrative labels include labels thatcan be directly observed or measured or indirectly observed or measured.Such labels include, but are not limited to, radiolabels that can bemeasured with radiation-counting devices; pigments, dyes or otherchromogens that can be visually observed or measured with aspectrophotometer; chemiluminescent labels that can be measured by aphotomultiplier-based instrument or photographic film, spin labels thatcan be measured with a spin label analyzer; and fluorescent moieties,where the output signal is generated by the excitation of a suitablemolecular adduct and that can be visualized by excitation with lightthat is absorbed by the dye or can be measured with standardfluorometers or imaging systems. The label can be a luminescentsubstance such as a phosphor or fluorogen; a bioluminescent substance; achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate or a spontaneously chemiluminescentproduct from a suitable precursor. The term label can also refer to a“tag” or hapten that can bind selectively to a labeled molecule suchthat the labeled molecule, when added subsequently, is used to generatea detectable signal.

Numerous labels are known by those of skill in the art and include, butare not limited to, microparticles, fluorescent dyes, haptens, enzymesand their chromogenic, fluorogenic and chemiluminescent substrates andother labels that are described in the Molecular Probes Handbook OfFluorescent Probes And Research Chemicals by Richard P. Haugland, 6thEd., (1996), and its subsequent 7th edition and 8th edition updatesissued on CD Rom in November 1999 and May 2001, respectively, thecontents of which are incorporated by reference, and in other publishedsources.

Exemplary fluorescent labels include, but are not limited to fluorescentdyes. As used herein, “fluorescent dye refers to non-protein moleculesthat absorb light and emit it at a longer wavelength. Exemplaryfluorescent dyes include, but are not limited to Alexa Fluor® dyes,fluorescein iso-thiocyanate (FITC), tetramethyl rhodamineiso-thiocyanate (TRITC), DyLight fluors, Cy dyes, IRDyes, HiLyte dyes,sulfonated and/or pegylated coumarin dyes, sulfonated and/or pegylatedxanthenes dyes, sulfonated or/pegylated cyanine dyes, and a sulfonatedand/or pegylated pyrene dyes.

An additional detectable label includes, but is not limited to DyomicsDY-631 NHS Ester. Other detectable labels that can be used include otherdyes, fluorophores, chromophores, mass tags, quantum dots and the like,and those disclosed in U.S. Pat. No. 6,924,372, which is incorporated byreference in its entirety.

Exemplary fluorescent labels also include, but are not limited to,biological fluorophores such as green fluorescent protein, and nanoscalecrystals such as quantum dots.

A further exemplary fluorescent label is available from Perkin Elmer aspart of the Pico Protein Reagent Kit (also referred to as the ProteinPico Assay Reagent Kit, part number 760498). In some embodiments, thefluorescent label comprises the Perkin Elmer Pico labeling dye. In someembodiments, labeling the protein of interest comprises adding a 4-20 μMPico dye solution to a sample comprising a protein of interest at aratio of about 1:1 by volume. In some embodiments, labeling the proteinof interest comprises adding a 4 μM, 5 μM, 6 μM, 10 μM, 12 μM, 14 μM, 15μM, 16 μM, 18 μM, 20 μM or 25 μM Pico dye solution to a samplecomprising a protein of interest. In some embodiments, the Pico dyesolution is added to the sample comprising the protein of interest at aratio of about 1:5 dye to sample by volume, 1:4 dye to sample by volume,1:3 dye to sample by volume, 1:2 dye to sample by volume 1:1 by volume,2:1 dye to sample by volume, 3:1 dye to sample by volume, 4:1 dye tosample by volume, or 5:1 dye to sample by volume. In some embodiments,labeling the protein of interest comprises adding a 16 μM Pico dyesolution to a sample comprising a protein of interest at a ratio ofabout 1:1 by volume. In some embodiments, the methods comprise andheating the sample and dye.

In some embodiments, the fluorescent label, or dye, is covalentlyattached to the protein of interest. In some embodiments, thefluorescent label comprises an amine-reactive group, and is covalentlyattached to free amines in the protein of interest. In some embodiments,the label is non-covalently attached to the protein of interest througha high-affinity interaction.

Additional suitable kits for protein labeling will be known to personsof ordinary skill in the art. Exemplary kits include, but are notlimited to, the Antibody/Protein Labeling Kit-FITC from MedChemExpress,and the (Fast) Alexa Fluor® Conjugation Kits.

Any covalently attached fluorescent label, and any methods of attachingthe fluorescent label are envisaged as within the scope of the instantmethods.

In some embodiments, the sample and the dye are heated to between about30° C. and 40° C. for about 5 to 40 minutes. In some embodiments, thesample and the dye are heated to between about 30° C. and 40° C. forabout 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes,about 25 minutes, about 30 minutes, about 35 minutes or about 40minutes. In some embodiments, the sample and the dye are heated to about35° C. for about 5 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 35 minutes or about40 minutes. In some embodiments, the sample and the label are heated toabout 35° C. for about 15 minutes. This heating step can produce asample comprising a denatured, labeled protein of interest. Excess labelcan optionally be removed from the sample, for example by using a spinfilter.

In some embodiments, the labeling reaction is stopped prior to thedeglycosylation reaction (quenching). For example, in those embodimentswhere the fluorescent label is the Perkin Elmer Pico labeling dye, thelabeling reaction can be stopped by adding an equal volume of PerkinElmer Pico stop buffer to the labeling reaction. In some embodiments thelabeling reaction is quenched by adding, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL,10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL or20 μL of an appropriate stop solution to the labeling reaction. In someembodiments, the dye is a Perkin Elmer Pico labeling dye, and thelabeling reaction is quenched by adding 5 μL of Perkin Elmer Pico stopsolution to the labeling reaction. Further exemplary stop buffers, forexample when the label comprises an amine-reactive fluorescent dye,include 1.5 M hydroxylamine, pH 8.5. The person of ordinary skill willbe able to select an appropriate stop buffer for various dye labelingreactions. Without wishing to be bound by theory, it is thought thatquenching the labeling reaction prevents labeling of the endoglycosidaseenzyme used for subsequent deglycosylation steps. This prevents orreduces a labeled endoglycosidase peak in the electropherogram used tovisualize the labeled sample.

Protein of Interest

The disclosure provides methods of preparing a sample comprising aprotein of interest for analysis using electrophoresis. In someembodiments, the protein of interest is glycosylated. In someembodiments, the methods comprise labeling the protein of interested,followed by deglycosylation.

All proteins of interest comprising post-translational modificationssuch as N-linked or O-linked glycosylation are envisaged as within thescope of the disclosure. In some embodiments, the protein of interest isa therapeutic protein, such as a therapeutic antibody, which can be adrug substance, a formulated drug substance or a drug product.

In some embodiments, the protein of interest comprises an antigenbinding domain. In some embodiments, the protein of interest is a fusionprotein. In some embodiments, the protein of interest comprisesantibody, an antibody fragment or a single chain-variable fragment(scFv). In some embodiments, the protein of interest is an antibody, anantibody fragment or an scFv.

In some embodiments, the protein of interest comprises a recombinanthuman protein. For example, the protein of interest can comprise a humanantibody or antibody fragment, or a humanized antibody or antibodyfragment.

As used herein “antibody” refers to an immunoglobulin moleculeconsisting of four polypeptide chains, two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chainhas a heavy chain variable region (HCVR or VH) and a heavy chainconstant region. The heavy chain constant region contains three domains,CH1, CH2 and CH3. Each light chain has a light chain variable region anda light chain constant region. The light chain constant region consistsof one domain (CL). The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term“antibody” includes reference to both glycosylated and non-glycosylatedimmunoglobulins of any isotype or subclass. The term “antibody” includesantibody molecules prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from a host celltransfected to express the antibody. The term antibody also includesbispecific antibody, which includes a heterotetrameric immunoglobulinthat can bind to more than one different epitope. Bispecific antibodiesare generally described in U.S. Pat. No. 8,586,713, which isincorporated by reference into this application.

The term “antigen-binding portion” of an antibody (or “antibodyfragment”), refers to one or more fragments of an antibody that retainthe ability to specifically bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature241:544-546), which consists of a VH domain, (vi) an isolated CDR, and(vii) an scFv, which consists of the two domains of the Fv fragment, VLand VH, joined by a synthetic linker to form a single protein chain inwhich the VL and VH regions pair to form monovalent molecules. Otherforms of single chain antibodies, such as diabodies are also encompassedunder the term “antibody” (see e.g., Holliger et al. (1993) PNAS USA90:6444-6448; Poljak et al. (1994) Structure 2:1 121-1 123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov et al. (1994) Mol. Immunol. 31: 1047-1058).Antibody portions, such as Fab and F(ab′)2 fragments, can be preparedfrom whole antibodies using conventional techniques, such as via papainor pepsin digestion of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques commonly known in the art (see Sambrook etal., 1989).

The term “human antibody” includes antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.

The term “humanized antibody”, as used herein, includes antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences, or otherwise modified to increase their similarity toantibody variants produced naturally in humans.

In some embodiments, the protein of interest is an antibody. In someembodiments, the antibody is selected from the group consisting of ananti-Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibody asdescribed in U.S. Pat. Appln. Pub. No. US2015/0203579A1), ananti-Programmed Cell Death Ligand-1 (e.g., an anti-PD-L1 antibody asdescribed in in U.S. Pat. Appln. Pub. No. US2015/0203580A1), ananti-D114 antibody, an anti-Angiopoetin-2 antibody (e.g., an anti-ANG2antibody as described in U.S. Pat. No. 9,402,898), ananti-Angiopoetin-Like 3 antibody (e.g., an anti-AngPt13 antibody asdescribed in U.S. Pat. No. 9,018,356), an anti-platelet derived growthfactor receptor antibody (e.g., an anti-PDGFR antibody as described inU.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti-ProlactinReceptor antibody (e.g., anti-PRLR antibody as described in U.S. Pat.No. 9,302,015), an anti-Complement 5 antibody (e.g., an anti-CS antibodyas described in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNFantibody, an anti-epidermal growth factor receptor antibody (e.g., ananti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or ananti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No.US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No.8,062,640 or 9,540,449), an Anti-Growth and Differentiation Factor-8antibody (e.g. an anti-GDF8 antibody, also known as anti-myostatinantibody, as described in U.S. Pat. No. 8,871,209 or 9,260,515), ananti-Glucagon Receptor (e.g. anti-GCGR antibody as described in U.S.Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1), ananti-VEGF antibody, an anti-IL1R antibody, an interleukin 4 receptorantibody (e.g., an anti-IL4R antibody as described in U.S. Pat. Appln.Pub. No. US2014/0271681A1 or U.S. Pat. No. 8,735,095 or 8,945,559), ananti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody asdescribed in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880), ananti-IL1 antibody, an anti-IL2 antibody, an anti-IL3 antibody, ananti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, ananti-IL7 antibody, an anti-interleukin 33 (e.g., anti-IL33 antibody asdescribed in U.S. Pat. No. 9,453,072 or 9,637,535), an anti-Respiratorysyncytial virus antibody (e.g., anti-RSV antibody as described in U.S.Pat. No. 9,447,173), an anti-Cluster of differentiation 3 (e.g., ananti-CD3 antibody, as described in U.S. Pat. Nos. 9,447,173 and9,447,173, and in U.S. Application No. 62/222,605), an anti-Cluster ofdifferentiation 20 (e.g., an anti-CD20 antibody as described in U.S.Pat. No. 9,657,102 and US20150266966A1, and in U.S. Pat. No. 7,879,984),an anti-CD19 antibody, an anti-CD28 antibody, an anti-Cluster ofDifferentiation-48 (e.g. anti-CD48 antibody as described in U.S. Pat.No. 9,228,014), an anti-Fel d1 antibody (e.g. as described in U.S. Pat.No. 9,079,948), an anti-Middle East Respiratory Syndrome virus (e.g. ananti-MERS antibody as described in U.S. Pat. Appln. Pub. No.US2015/0337029A1), an anti-Ebola virus antibody (e.g. as described inU.S. Pat. Appln. Pub. No. US2016/0215040), an anti-Zika virus antibody,an anti-Lymphocyte Activation Gene 3 antibody (e.g. an anti-LAG3antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factorantibody (e.g. an anti-NGF antibody as described in U.S. Pat. Appln.Pub. No. US2016/0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) andan anti-Protein Y antibody. In some embodiments, the bispecific antibodyis selected from the group consisting of an anti-CD3× anti-CD20bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos.US2014/0088295A1 and US20150266966A1), an anti-CD3× anti-Mucin 16bispecific antibody (e.g., an anti-CD3× anti-Muc16 bispecific antibody),and an anti-CD3× anti-Prostate-specific membrane antigen bispecificantibody (e.g., an anti-CD3× anti-PSMA bispecific antibody).

In some embodiments, the protein of interest is selected from the groupconsisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab,alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab,benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximabvedotin, brodalumab, canakinumab, capromab pendetide, certolizumabpegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab,durvalumab, eculizumab, elotuzumab, emicizumab-kxwh,emtansinealirocumab, evinacumab, evolocumab, fasinumab, golimumab,guselkumab, ibritumomab tiuxetan, idarucizumab, infliximab,infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab,necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab,ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab,pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab,reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab,tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, andvedolizumab.

Proteins of interest can be created or isolated by any means known inthe art. These include recombinant means, such as proteins (e.g.antibodies) expressed using a recombinant expression vector transfectedinto a host cell. Antibodies that are proteins of interest can beisolated from a recombinant, combinatorial human antibody library,isolated from an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or prepared, expressed, created or isolated by any othermeans that involves splicing of human immunoglobulin gene sequences toother DNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, recombinant human antibodies are subjected to invitro mutagenesis (or, when an animal transgenic for human Ig sequencesis used, in vivo somatic mutagenesis) and thus the amino acid sequencesof the VH and VL regions of the recombinant antibodies are sequencesthat, while derived from and related to human germline VH and VLsequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

In some embodiments, the protein of interest comprises an fragmentcrystallizable (Fc) domain. For example, the protein of interest can bea receptor-Fc-fusion protein or a soluble TCR-Fc fusion protein. In someembodiments, the receptor-Fc-fusion protein is a trap protein.

Fusion proteins comprise two or more parts of the protein which are nototherwise found together in nature. For example, an “Fc fusion protein”can comprise an Fc portion of an immunoglobulin molecule, which is fusedto another heterologous domain, such as a receptor ligand bindingdomain. Preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.,Proc. Natl. Acad. ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677,1990; and Hollenbaugh et al., “Construction of Immunoglobulin FusionProteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11, 1992. “Receptor Fc fusion proteins” comprise one ormore extracellular domain(s) of a receptor coupled to an Fc moiety,which in some embodiments comprises a hinge region followed by a CH2 andCH3 domain of an immunoglobulin. In some embodiments, the Fc-fusionprotein contains two or more distinct receptor chains that bind to a oneor more ligand(s). For example, an Fc-fusion protein is a trap, such asfor example an interleukin 1 (IL-1) trap (e.g., rilonacept, whichcontains the IL-1 RAcP ligand binding region fused to the IL-1 R1extracellular region fused to Fc of hlgG1; see U.S. Pat. No. 6,927,004),or a vascular endothelial growth factor A (VEGF) trap (e.g.,aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hlgG1;see U.S. Pat. Nos. 7,087,411 and 7,279,159).

In some embodiments, the protein of interest is a fusion protein, suchas a receptor fusion protein. Receptor fusion proteins can include,intera alia, trap proteins and mini trap proteins.

The term “fusion protein” refers to a molecule comprising two or moreproteins or fragments thereof linked by a covalent bond via theirindividual peptide backbones, optionally generated through geneticexpression of a polynucleotide molecule encoding the fusion protein.

In some embodiments, the protein of interest is a trap protein or a minitrap protein. In some embodiments, trap proteins are engineeredtherapeutic proteins capable of acting as decoy receptors to bind to andantagonize or modulate the activity of a target protein. An exemplarytrap protein comprises one or more receptor components that mimic thebinding domain of the receptor for its target protein (e.g., the VEGFreceptor Ig domain 2 of Flt-1 and the Ig domain 3) fused to a human IgGconstant region, optionally including additional domains such aslinkers, dimerization or multimerization domains, and cleavage sites. Insome embodiments, the trap protein is truncated or of reduced size (amini trap), for example through protein cleavage, which can aid intissue penetration of the mini trap. Non-limiting examples of trapproteins include an IL-1 trap (e.g., rilonacept, which contains theIL-1RAcP ligand binding region fused to the IL-1R1 extracellular regionwhich in turn is fused to the Fc of hlgG1) (e.g., SEQ ID NO: 1) (seeU.S. Pat. No. 6,927,004), or a VEGF trap (e.g., aflibercept, whichcontains the Ig domain 2 of the VEGF receptor Flt1 fused to the Igdomain 3 of the VEGF receptor Flk1 which in turn is fused to Fc ofhlgG1. See, e.g., U.S. Pat. Nos. 7,087,411, 7,279,159; see also U.S.Pat. No. 5,610,279 for etanercept (TNF trap), the contents of each ofwhich are incorporated by reference in their entirety herein.

Protein Production

The protein of interest assayed by the methods described herein can beproduced by any method known in the art. For example, the protein ofinterest can be produced by cell cultures. The cell cultures can be a“fed-batch cell culture” or “fed-batch culture” which refers to a batchculture wherein the cells and culture medium are supplied to theculturing vessel initially and additional culture nutrients are slowlyfed, in discrete increments, to the culture during culturing, with orwithout periodic cell and/or product harvest before termination ofculture. Fed-batch culture includes “semi-continuous fed-batch culture”wherein periodically whole culture (which may include cells and medium)is removed and replaced by fresh medium. Fed-batch culture isdistinguished from simple “batch culture” whereas all components forcell culturing (including the animal cells and all culture nutrients)are supplied to the culturing vessel at the start of the culturingprocess in batch culture. Fed-batch culture may be different from“perfusion culture” insofar as the supernatant is not removed from theculturing vessel during a standard fed-batch process, whereas inperfusion culturing, the cells are restrained in the culture by, e.g.,filtration, and the culture medium is continuously or intermittentlyintroduced and removed from the culturing vessel. However, removal ofsamples for testing purposes during fed-batch cell culture iscontemplated. The fed-batch process continues until it is determinedthat maximum working volume and/or protein production is reached, andprotein is subsequently harvested.

The cell culture can be a “continuous cell culture” which is a techniqueused to grow cells continually, usually in a particular growth phase.For example, if a constant supply of cells is required, or theproduction of a particular protein of interest is required, the cellculture may require maintenance in a particular phase of growth. Thus,the conditions must be continually monitored and adjusted accordingly inorder to maintain the cells in that particular phase.

The cells are cultured in cell culture medium. The terms “cell culturemedium” and “culture medium” refer to a nutrient solution used forgrowing mammalian cells that typically provides the necessary nutrientsto enhance growth of the cells, such as a carbohydrate energy source,essential (e.g., phenylalanine, valine, threonine, tryptophan,methionine, leucine, isoleucine, lysine, and histidine) and nonessential(e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, proline, serine, and tyrosine) amino acids, traceelements, energy sources, lipids, vitamins, etc. Cell culture medium maycontain extracts, e.g., serum or peptones (hydrolysates), which supplyraw materials that support cell growth. Media may contain yeast-derivedor soy extracts, instead of animal-derived extracts. Chemically definedmedium refers to a cell culture medium in which all of the chemicalcomponents are known (i.e., have a known chemical structure). Chemicallydefined medium is entirely free of animal-derived components, such asserum- or animal-derived peptones. In one embodiment, the medium is achemically defined medium.

A “cell line” refers to a cell or cells that are derived from aparticular lineage through serial passaging or subculturing of cells.The term “cells” is used interchangeably with “cell population.” Theterm “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes, such as bacterial cells, mammalian cells, human cells,non-human animal cells, avian cells, insect cells, yeast cells, or cellfusions such as, for example, hybridomas or quadromas. In certainembodiments, the cell is a human, monkey, ape, hamster, rat or mousecell. In other embodiments, the cell is selected from the followingcells: Chinese Hamster Ovary (CHO) (e.g., CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5,Colo25, HB 8065, HL-60, lymphocyte, e.g., Jurkat (T lymphocyte) or Daudi(B lymphocyte), A431 (epidermal), U937, 3T3, L cell, C127 cell, SP2/0,NS-0, MMT cell, stem cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, the cell comprises one or moreviral genes, e.g., a retinal cell that expresses a viral gene (e.g., aPER.C6® cell). In some embodiments, the cell is a CHO cell. In otherembodiments, the cell is a CHO K1 cell.

Cells can be transformed with heterologous polynucleotides encoding aprotein of interest using any method known in the art, including, butnot limited to, transformation, transfection, electroporation, and thelike.

The term “heterologous polynucleotide” refers a polynucleotide sequenceencoding a heterologous nucleotide sequence not found in the wild typecell, which can include a sequence encoding the protein of interest.Exemplary heterologous polynucleotides include vectors comprising asequence encoding the protein of interest, including, but not limitedto, plasmid, phage and viral particles. Optionally, the vector allowstransfer of a particular nucleic acid molecule to a cell. Whenintroduced into an appropriate cell, an expression vector contains thenecessary genetic elements to direct expression of the protein ofinterest. Exemplary vectors can include transcriptional promoterelements (i.e., an expression control sequence), which are operativelylinked to the sequence encoding the protein of interest. The vector maybe composed of either DNA, or RNA, or a combination of the two (e.g., aDNA-RNA chimera). Optionally, the vector may include a polyadenylationsequence, one or more restriction sites as well as one or moreselectable markers such as phosphotransferase or hygromycinphosphotransferase. Additionally, depending on the cell type chosen andthe vector employed, other genetic elements such as an origin ofreplication, additional nucleic acid restriction sites, enhancers, andsequences conferring inducibility of transcription, may also beincorporated into the vector. Selection of appropriate vectors andtransformation methods will be apparent to those of ordinary skill inthe art.

Glycosylation

In some embodiments, the protein of interest is glycosylated. Theglycosylation can included N-linked glycosylation, O-linkedglycosylation or a combination thereof. Many proteins and polypeptidesof interest produced in cell culture are glycoproteins that containcovalently linked carbohydrate structures including oligosaccharidechains (glycans). These oligosaccharide chains are linked to the proteinin the endoplasmic reticulum and the Golgi apparatus via eitherN-linkages or O-linkages. The oligosaccharide chains may comprise asignificant portion of the mass of the glycoprotein. The oligosaccharidechains can play roles including facilitating correct folding of theglycoprotein, mediating protein-protein interactions, conferringstability, conferring advantageous pharmacodynamic and/orpharmacokinetic properties, inhibiting proteolytic digestion, targetingthe glycoprotein to the proper secretory pathway and targeting theglycoprotein to a particular organ or organs.

In some embodiments, the protein of interest comprises N-linkedglycosylation. Generally, N-linked oligosaccharide chains are added tothe nascent, translocating protein in the lumen of the endoplasmicreticulum. The oligosaccharide is added to the amino group on the sidechain of an asparagine residue contained within a target consensussequence such as Asn-X-Ser/Thr or in some instances Asn-X-Cys, where Xmay be any amino acid except proline. The initial oligosaccharide chainis usually trimmed by specific glycosidase enzymes in the endoplasmicreticulum, resulting in a short, branched core oligosaccharide composedof two N-acetylglucosamine and three mannose residues.

After initial processing in the endoplasmic reticulum, the glycoproteinmay undergo further processing before being secreted to the cellsurface. N-linked oligosaccharide chains may be modified by the additionof mannose residues, resulting in a high-mannose oligosaccharide.Alternatively, one or more monosaccharides units of N-acetylglucosaminemay be added to the core mannose subunits to form complexoligosaccharides. Galactose may be added to the N-acetylglucosaminesubunits, and sialic acid subunits may be added to the galactosesubunits, resulting in chains that terminate with either a sialic acid,a galactose or an N-acetylglucosamine residue. Additionally, a fucoseresidue may be added to an N-acetylglucosamine residue of the coreoligosaccharide. Each of these additions is catalyzed by specificglycosyl transferases.

In addition to being modified by the N-linked glycosylation pathway,glycoproteins may also be modified by the addition of O-linkedoligosaccharide chains to specific serine or threonine residues as theyare processed in the Golgi apparatus. The residues of an O-linkedoligosaccharide are added one at a time and the addition of each residueis catalyzed by a specific enzyme. In contrast to N-linkedglycosylation, the consensus amino acid sequence for O-linkedglycosylation is less well defined. In some embodiments, the protein ofinterest comprises O-linked glycosylation. In some embodiments, theO-linked glycosylation comprises the attachment of a sugar molecular toa serine (Ser) or Threonine (Thr) amino acid of the protein of interest.

In some embodiments, the protein of interest is a glycosylated protein.In some embodiments the glycosylated protein comprises at least oneattached glycan. In some embodiments the protein of interest comprisesat least 1 attached glycan, at least 2 attached glycans, at least 3attached glycans, at least 4 attached glycans, at least 5 attachedglycans, at least 6 attached glycans, at least 7 attached glycans, atleast 8 attached glycans, at least 9 attached glycans, at least 10attached glycans, at least 11 attached glycans, at least 12 attachedglycans, at least 15 attached glycans, at least 20 attached glycans orat least 25 attached glycans. In some embodiments, the protein ofinterest has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 attached glycans. In some embodiments,the glycans are N-linked. In some embodiments, the glycans are O-linked.In some embodiments, the protein of interest comprises both N- andO-linked glycans.

In some embodiments, the protein of interest is a glycosylated protein.In some embodiments, the protein of interest comprises at least oneglycosylation site. In some embodiments, the protein of interestcomprises at least one glycosylation site, at least two glycosylationsites, at least 3 glycosylation sites, at least 4 glycosylation sites,at least 5 glycosylation sites, at least 6 glycosylation sites, at least7 glycosylation sites, at least 8 glycosylation sites, at least 9glycosylation sites, at least 10 glycosylation sites, at least 10glycosylation sites, at least 11 glycosylation sites, at least 12glycosylation sites, at least 15 glycosylation sites, at least 20glycosylation sites or at least 25 glycosylation sites. In someembodiments, the at least glycosylation site is an N-linkedglycosylation site, for example an asparagine within the N-linkedglycosylation consensus sequence. In some embodiments, the at least oneglycosylation site is an O-linked glycosylation site, for example aserine or threonine. In some embodiments, the protein of interestcomprises both at least one N-linked glycosylation site and at least oneO-linked glycosylation site.

In some embodiments, glycans comprise at least at least 0.5%, 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65% or at least 75% of the total weight of the glycosylated protein (5%weight/weight, or w/w). In some embodiments, glycans comprise at least5% of the total weight of the glycosylated protein (5% w/w). In someembodiments, glycans comprise at least 10% of the total weight of theglycosylated protein (10% w/w). Methods of determining the percentage ofprotein weight made up of glycans will be readily apparent to one ofordinary skill in the art and include, but are not limited to, comparingexpected weight derived from amino acid sequence to actual weightdetermined by the electrophoresis analysis methods described herein.

Reference Standards

In some embodiments, a reference standard is subjected to the samemethods of preparation in parallel to the sample comprising the proteinof interest, and analyzed in parallel to the sample comprising theprotein of interest. In some embodiments, methods comprise comparing oneor more characteristics of the protein of interest to the referencestandard. For example, the methods can include comparingelectropherograms of the protein of interest and the reference standard.

As used herein, a “reference standard” refers to a sample comprising aprotein that has previously been analyzed using the methods known in theart, and whose characteristics are known. Known characteristics can bedetermined from the amino acid sequence of the reference standard (e.g.,predicted molecular weight), or experimentally determined (e.g.,electropherogram profile). These characteristics can include, but arenot limited to, expected and experimentally determined molecular weight,electropherogram(s) generated using the methods described herein orknown methods in the art, isoelectric point, extinction coefficient (ameasure of how strongly the protein of interest absorbs light at a givenwavelength), number of glycosylation sites, and molecular weight ofattached glycans. A reference standard may be similar in one or morecharacteristics to a protein of interest. For example, both the proteinof interest and the reference standard may be monoclonal antibodies, orcomprise an Fc domain, be of similar molecular weight, or the like.

In some embodiments, the reference standard comprises the protein ofinterest. For example, the reference standard can be from a separatebatch of the protein of interest than the sample, which has beenpreviously characterized and stored under controlled conditions toprevent degradation.

In some embodiments, the disclosure provides methods of preparing asample comprising a protein of interest and a reference standard foranalysis using electrophoresis, comprising (a) denaturing the sample andthe reference standard; (b) labeling the protein of interest and thereference standard with a fluorescent label to produce a labeled sampleand labeled reference standard; (c) quenching the labeling reaction s ofthe protein of interest and the reference standard, (d) deglycosylatingthe labeled sample and labeled reference standard with anendoglycosidase; and (e) performing electrophoresis on the labeledsample and labeled reference standard; wherein the sample and thereference standard are labeled and quenched in steps (b) and (c) priorto deglycosylation in step (d).

In some embodiments, the electrophoresis is microchip capillaryelectrophoresis (MCE), and the output is an electropherogram. In someembodiments, the methods comprise determining a main peak intensity forthe protein of interest and the reference standard, and comparing theintensity values of the main peak for the protein of interest and themain peak for the reference standard. In some embodiments, the main peakof the protein of interest or the reference standard is glycosylated. Insome embodiments, the main peak of the protein of interest or thereference standard is not glycosylated, i.e., has been deglycosylatedafter labeling using the methods described herein. In some embodiments,determining the main peak comprises determining the height of the mainpeak. In some embodiments, determining the main peak comprisesdetermining the area of the main peak. In some embodiments, determiningthe main peak comprises determining the time corrected area of the mainpeak, which is the peak area divided by its migration time. In someembodiments, the main peak intensity of the protein of interest iswithin 50% to 150%, 50% to 140%, 50% to 130%, 50% to 120%, 50% to 110%,50% to 100%, 50% to 90%, 60% to 150%, 70% to 150%, 80% to 150%, 90% to150%, 100% to 150%, 110% to 150%, 120% to 150%, 130% to 150%, 140% to150%, 60% to 140%, 70% to 140%, 70% to 130%, 70% to 120%, 70% to 110%,80% to 140%, 80% to 130%, 80% to 120%, 80% to 110%, 80% to 100%, 90% to140%, 90% to 130%, 90% to 120%, 90% to 110% or 90% to 100% of the mainpeak intensity of the reference standard. In some embodiments, the mainpeak intensity of the protein of interest is within 60% to 140%, 70% to130%, 80% to 120%, or 90% to 110% of the main peak intensity of thereference standard. In some embodiments, the main peak intensity of theprotein of interest is within 70% to 130% of the main peak intensity ofthe reference standard. Determining main peak intensity of the proteinof interest relative to the reference standard can ensure properseparation by the CE or MCE instrument, and data quality.

Electrophoresis

Provided herein are methods of analyzing a sample comprising a proteinof interest prepared using the methods described herein usingelectrophoresis.

Electrophoresis based methods for analyzing proteins include, but arenot limited to, gel-based methods such as sodium dodecyl (lauryl)sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE),polyacrylamide gel electrophoresis in the presence of lithium dodecylsulfate, free-flow electrophoresis, isoelectric focusing, capillary gelelectrophoresis, capillary electrophoresis (CE) and microchip capillaryelectrophoresis (MCE).

In some embodiments, the electrophoresis is CE. In some embodiments, theCE comprises a lithium dodecyl sulfate (LDS) buffer.

In some embodiments, the electrophoresis is MCE. The terms “MCE” or“Microchip Capillary Electrophoresis” and “capillary electrophoresis(CE)” refer to capillary electrophoresis (CE) and its microfluidiccounterpart (MCE), which are used to separate analytes in a sample. MCEtechniques can be used to separate, identify, and quantify proteins ofinterest, impurities in the protein sample, and analyze breakdownproducts of the protein of interest such as protein fragments. CE andMCE separate analytes based on electrophoretic mobility when a voltageis applied to a sample. The presence of gel matrix (e.g., gelelectrophoresis) will separate analytes based on size as well as charge.Impurities in the sample include, but are not limited to proteinaggregates, protein fragments, protein multimers, and assaycontaminants.

In MCE, the denatured labeled protein of interest is diluted andsubjected to MCE to separate the diluted protein sample on a microchipcapillary electrophoresis system to produce an electropherogram. Becausemultiple samples can be run in parallel on the same microchip, MCE basedmethods are readily adaptable to high throughput approaches. Further,MCE is rapid, and uses minimal sample volume.

As used herein, an electropherogram is a plot that results fromelectrophoretic methods such as CE or MCE. The electropherogram containspeaks corresponding to the protein of interest and impurities.

Methods of analyzing electropherograms are known in the art, and includecomparing the position, size and areas under individual peaks. Methodsof calculating peak area for an electropherogram (area under the peak)are known in the art, and include, for example, integrating to estimatethe area under a peak. Peak area can be calculated using software suchas Empower.

Instrumentation for conducting the disclosed MCE assays is commerciallyavailable. In some embodiments, the disclosed MCE assays are performedusing LabChip GXII, LabChip GXII Touch™, LabChip GXII Touch™ HT and aProtein Express Assay LabChip (LabChip® HT Protein Express Chip).

Instrumentation for conducting the disclosed CE assays is alsocommercially available. For example, CE assays can be performed using aBeckman Coulter capillary electrophoresis system such as the PA 800 PlusPharmaceutical Analysis System.

In some embodiments of the methods described herein, the methods furthercomprise labeling and running a protein standard molecular weight ladderto assess the size of the protein of interest. Protein molecular weightladders will be known to persons of ordinary skill in the art, andinclude PageRuler, Mark12, BenchMark, PageRuler High Range and PageRulerLow Range available from ThermoFisher, as well as the HT PICO ProteinExpress ladder from the Protein Pico Assay Reagent Kit from PerkinElmer.Selection of appropriate ladder based on size of the protein of interestwill be apparent to one of ordinary skill in the art.

Applications

The disclosure provides methods of characterizing a protein of interest,using the methods of labeling, deglycosylation and electrophoresisdescribed herein.

Analyzing a protein of interest can include, but is not limited to,characterizing the number, position, height, width, intensity, size orarea of one or more peaks in an electropherogram generated by CE or MCE.

Characterizing the number and position of peaks in an electropherogramcan determine whether or not degradation products of the protein ofinterest are present in the sample, for example as peaks with molecularweights that are less than that of the main peak. Comparison of peaksgenerated from non-deglycosylated protein of interest, and protein ofinterest deglycosylated and labeled using the methods described herein,can determine whether or not glycosylated forms of the protein ofinterest are present in the sample, as a deglycosylated main peak of theprotein of interest will have a lower molecular weight than aglycosylated main peak of a protein of interest.

The methods of the instant disclosure can be used to assay the stabilityof proteins of interest under various conditions. These include storageconditions for protein of interest that has been formulated as a drugsubstance or drug product. Comparisons of peak number and peak area, forexample between a reference sample comprising a protein of interest anda stressed sample thereof, can be used to determine the stability of theprotein of interest over time, and under various conditions such as highor low pH, or exposure to light.

Accordingly, the disclosure provides methods of determining stability ofa protein of interest using the methods of labeling and deglycosylatinga protein of interest described herein. In some embodiments, the methodscomprise (a) stressing a sample comprising the protein of interest; (b)denaturing the stressed sample and a non-stressed sample comprising theprotein of interest; (c) labeling the protein of interest in thestressed sample and the non-stressed sample with a fluorescent label toproduce a labeled stressed sample and a labeled non-stressed sample; (d)quenching un-reacted fluorescent label in the labeled stressed sampleand the labeled non-stressed sample; (e) deglycosylating the labeledstressed sample and the labeled non-stressed sample with anendoglycosidase; (f) performing microchip capillary electrophoresis(MCE) on the labeled stressed sample and the labeled non-stressed sampleto generate electropherograms for the stressed sample and thenon-stressed sample; and (g) comparing the electropherograms from thestressed sample and the nonstressed sample; wherein the stressed sampleand the non-stressed sample are labeled and quenched in steps (c) and(d) prior to deglycosylation in step (e).

Any methods of stressing a protein of interest in a sample are envisagedas within the methods of the disclosure, including, but not limited to,chemicals, pH, radiation, light, freeze-thaw cycles, lyophilization andheat.

In some embodiments, stressing the sample comprising the protein ofinterest comprises thermally stressing the sample. Thermally stressingthe sample can include simulating storage conditions for protein ofinterest formulated as drug substance or formulated drug product, i.e.the stressed sample is held at about −80° C. to −30° C. or about 2° C.to about 8° C., respectively. In other embodiments, thermally stressingthe sample comprises simulating handling and transport conditions forthe sample. In other embodiments, thermally stressing the samplecomprises inducing forced degradation of the sample, for example byincreasing the temperature to which the sample is exposed.

In some embodiments, stressing the sample comprising the protein ofinterest comprises thermally stressing the sample. In some embodiments,the thermal stress comprises holding the sample between 25° C. and 45°C. In some embodiments, the thermal stress comprises holding the sampleat 2° C., 4° C., 6° C., 8° C., 10° C., 12° C., 14° C., 16° C., 18° C.,20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 35° C., 37° C.or 40° C. In some embodiments, the thermal stress comprises holding thesample at 37° C. In some embodiments, the thermal stress comprisesholding the sample at 22° C. to 26° C. In some embodiments, the thermalstress comprises holding the sample at 30° C. In some embodiments, thethermal stress comprising holding the protein at between about 25° C.and 45° C. In some embodiments, the thermal stress comprises holding thestressed sample for at least 1 week, 2 weeks, 3 weeks 4 weeks, 5 weeks,6 weeks, 7 weeks, 8 weeks 9 weeks, 10 weeks, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months orone year. In some embodiments, the stressed sample is held for 2 weeks.In some embodiments, the stressed sample is held for 4 weeks.

In some embodiments, thermally stressing the sample comprises holdingthe sample at between about 25° C. and about 45° C. for at least 1 week,at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks,at least 6 weeks, at least 7 weeks or at least 8 weeks. In someembodiments, thermally stressing the sample comprises holding the sampleat between about 30° C. and about 45° C. for at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks or at least 8 weeks.

In some embodiments, stressing the sample comprises at least onefreeze/thaw cycle. For example, starting from a liquid sample, loweringthe temperature until the sample freezes, and then returning the sampleto a temperature where it is a liquid prior to analysis.

In some embodiments, stressing the sample comprises exposing the sampleto storage conditions. In some embodiments, the storage conditionscomprise a temperature of about −80° C. to −30° C. for at least 1 week,at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months,at least 3 months, at least 6 months, at least 8 months, at least 12months, at least 18 months, at least 24 months or at least 30 months. Insome embodiments, the storage conditions comprise a temperature of about2° C. to 8° C. for at least 1 week, at least 2 weeks, at least 3 weeks,at least 1 month, at least 2 months, at least 3 months, at least 6months, at least 8 months, at least 12 months or at least 18 months.

In some embodiments, stressing the sample comprises mechanicallyagitating the sample, for example using a Vortex or magnetic stirrer.

In some embodiments, stressing the sample comprises lyophilizing andrehydrating the sample. Methods of lyophilizing a sample comprising aprotein of interest will be known to persons of ordinary skill in theart and include, for example freeze drying and spray drying.

In some embodiments, stressing the sample comprises exposing the sampleto light, radiation, singlet oxygen species, free radicals, high pHconditions or low pH conditions. Exemplary low pH conditions include,inter alia, exposing the sample to a pH of less than 7.0, for example apH of less than 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.0, 1.5 or 1.0.Exemplary high pH conditions include, inter alia, exposing the sample toa pH of greater than 7.0, for example a pH of greater than 8.0, 8.5,9.0, 9.5 or 10.0.

In some embodiments, stressing the sample comprises exposing the sampleto light. Exposure to light can include light of any wavelength, or anyrange of wavelengths. In exemplary embodiments, samples are expose tocool white fluorescent light or near ultraviolet light. Exemplary coolwhite fluorescent light comprises light of mixed wavelengths that has acorrelated color temperature (CCT) of about 4,100 to about 4,500 kelvins(K). In some aspects, the cool white fluorescent light has a CCT of4,100K. In some aspects, exposing the sample to light comprises exposingthe sample to about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3.0 million lux hours accumulated exposure of cool white light.In some aspects, exposing the sample to light comprises exposing thesample to about 1.2 or about 2.4 million lux hours accumulated exposureof cool white light. Exemplary near ultraviolet light has a wavelengthof about 300 nm to about 400 nm. In some aspects, the near ultravioletlight has an integrated energy of between about 100 watt hours/squaremeter to about 600 watt hours/square meter. In some aspects, the nearultraviolet light has an integrated energy of about 100, 200, 300, 400,500 or 600 watt hours/square meter.

A reduction in main peak area between the reference sample and thestressed version thereof can, for example, indicate a reduction ofprotein of interest in the main peak through degradation. In someembodiments, the area of the main peak of the stressed protein ofinterest is reduced by at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 6 at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35% or at least 40% compared to the main peak of the non-stressedprotein of interest. Similarly, an increase in the area of low molecularweight peaks in the stressed reference sample compared to the referencesample can indicate degradation of the protein of interest, as theabundance of the lower molecular weight species representing degradationproducts of the protein of interest increases. In some embodiments, thearea of at least one low molecular weight peak of the stressed proteinof interest is increased by at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6 at least 7%, at least 8%, at least 9%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35% or at least 40% compared to at least one low molecular weightpeak of the non-stressed protein of interest.

Kits and Articles of Manufacture

The disclosure provides kits including one or more the disclosedbuffers, enzymes, dyes and reference standards used in the methods ofdeglycosylation and labeling described herein. The kits can include acontainer for the ingredients. The buffers can be in solution or inlyophilized form. In some embodiments, the kits include a secondcontainer containing a diluent or reconstituting solution for thelyophilized formulation; and optionally, instructions for the use of thesolution or the reconstitution and/or use of the lyophilized buffers orpowdered ingredients.

The kits described herein may further include additional reagents neededto perform the disclosed MCE assays including one or more of a buffer, adiluent, and a filter. The buffer and reagents can be in a bottle, avial, or test tube.

In some embodiments, the kits include instructions for use.

The present description sets forth numerous exemplary configurations,methods, parameters, and the like. It should be recognized, however,that such description is not intended as a limitation on the scope ofthe present disclosure, but is instead provided as a description ofexemplary embodiments.

ENUMERATED EMBODIMENTS

The invention may be defined by reference to the following enumerated,illustrative embodiments:

1. A method of analyzing a sample comprising a protein of interest, themethod comprising:

a. denaturing the sample;

b. labeling the sample with a fluorescent label to produce a labeledsample;

c. quenching un-reacted fluorescent label in the labeled sample;

d. deglycosylating the labeled sample with an endoglycosidase; and

e. performing electrophoresis on the labeled sample;

wherein the sample is denatured, labeled and quenched in steps (a)through (c) prior to deglycoslation in step (d).

2. The method of embodiment 1, wherein the protein of interest comprisesat least one glycosylation site.

3. The method of embodiment 1 or 2, wherein the protein is of interestis a glycosylated protein.

4. The method of embodiment 3, wherein the glycosylated proteincomprises at least one attached glycan.

5. The method of any one of embodiments 1-4, wherein the protein ofinterest comprises an antigen binding domain.

6. The method of embodiment 5, wherein the protein of interest comprisesan antibody, an antibody fragment or an scFv.

7. The method of any one of embodiments 1-6, wherein the protein ofinterest comprises an Fc domain.

8. The method of any one of embodiments 1-7, wherein the protein ofinterest comprises a receptor fusion protein.

9. The method of embodiment 8, wherein the receptor fusion protein is areceptor-Fc-fusion protein or a soluble TCR-Fc fusion protein.

10. The method of embodiment 8 or 9, wherein the receptor fusion proteinis a trap protein or a mini trap protein.

11. The method of any one of embodiments 1-10, wherein the protein ofinterest is a recombinant human protein.

12. The method of any one of embodiments 2-11, wherein the glycosylationsite comprises an Asn-X-Ser/Thr consensus sequence.

13. The method of any one of embodiments 4-12, wherein the at least oneattached glycan is N-linked.

14. The method of embodiment 13, wherein the at least one attachedglycan is N-linked to an asparagine in the glycosylated protein.

15. The method of any one of embodiments 1-14, wherein theendoglycosidase catalyzes deglycosylation of N-linked glycans.

16. The method of embodiment any one of embodiments 1-15, wherein theendoglycosidase is selected from the group consisting ofPeptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H),Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1,Endoglycosidase F2 and Endoglycosidase F4.

17. The method of any one of embodiments 1-15, wherein theendoglycosidase is PNGase F.

18. The method of embodiment 17, wherein the PNGase F is Rapid PNGase F.

19. The method of embodiment 18, wherein the Rapid PNGase F isnon-reducing.

20. The method of any one of embodiments 17-19, wherein deglycosylatingthe sample comprises heating the sample to about 50° C. for 10 minutes.

21. The method of any one of embodiments 1-20, wherein deglycosylatingthe sample comprises a reaction mixture comprising between 0.2-1.5 mglabeled protein of interest, and between 1-5 μL Rapid PNGase F in a 10μL reaction volume, excluding the volume of the Rapid PNGase F.

22. The method of embodiment 21, wherein the reaction mixture comprises0.2 mg labeled protein of interest.

23. The method of embodiment 21, wherein the reaction mixture comprises5 μL Rapid PNGase F.

24. The method of any one of embodiments 21-23, wherein the reactionmixture comprises a buffer.

25. The method of any one of embodiments 4-11, wherein the at least oneglycan is an O-linked glycan.

26. The method of embodiment 25, wherein the endoglycosidase catalyzesdeglycosylation of O-linked glycans.

27. The method of embodiment 25 or 26, wherein the endoglycosidasecomprises Endo-α-N-acetylgalactosamindase (O-glycosidase).

28. The method of any one of embodiments 1-27, wherein labeling thesample with the fluorescent label comprises heating the sample to about35° C. for 10-30 minutes.

29. The method of any one of embodiments 1-27, wherein labeling thesample with the fluorescent label comprises heating the sample to about35° C. for about 15 minutes.

30. The method of any one of embodiments 1-29, wherein the sample isdenatured using a reducing solution.

31. The method of embodiment 30, wherein the reducing solution comprisesdithiothreitol (DTT).

32. The method of any one of embodiments 1-29, wherein the sample isdenatured using a non-reducing solution.

33. The method of embodiment 32, wherein the non-reducing solutioncomprises iodoacetamide (IAM).

34. The methods of any one of embodiments 1-33, wherein denaturing thesample comprises heating the sample to between 40° C. and 99° C. forbetween 1 minute and 5 hours.

35. The methods of any one of embodiments 1-33, wherein denaturing thesample comprises heating the sample to between 50° C. and 99° C. forbetween 1 to 60 minutes.

36. The method of any one of embodiments 1-35, wherein quenching theun-reacted fluorescent label comprises adding a stop solution.

37. The method of any one of embodiments 1-36, further comprisinganalyzing a reference standard in parallel to the sample.

38. The method of any one of embodiments 1-37, wherein theelectrophoresis is selected from the group consisting of gelelectrophoresis, isoelectric focusing, capillary electrophoresis (CE) ormicrochip capillary electrophoresis (MCE).

39. The method of any one of embodiments 1-37, wherein theelectrophoresis is MCE.

40. The method of embodiment 39, wherein the MCE is carried out using anMCE instrument.

41. The method of any one of embodiments 1-40, wherein method results inreduced free dye interference in the less than 20 kDa range and areduced or absent endoglycosidase peak in an electropherogram whencompared to an electropherogram generated using a sample labeled afterdeglycosylation.

42. The method of embodiment 41, wherein the endoglycosidase peak isreduced by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% when compared to an electropherogram generated using a samplelabeled after deglycosylation.

43. The method of embodiment 41, wherein the endoglycosidase peak isabsent in an electropherogram when compared to an electropherogramgenerated using a sample labeled after deglycosylation.

44. A method of determining stability of a protein of interestcomprising:

a. stressing a sample comprising the protein of interest;

b. denaturing the stressed sample and a non-stressed sample comprisingthe protein of interest;

c. labeling the stressed sample and the non-stressed sample with afluorescent label to produce a labeled stressed sample and a labelednon-stressed sample;

d. quenching un-reacted fluorescent label in the labeled stressed sampleand the labeled non-stressed sample;

e. deglycosylating the labeled stressed sample and the labelednon-stressed sample with an endoglycosidase;

f. performing microchip capillary electrophoresis (MCE) on the labeledstressed sample and the labeled non-stressed sample to generateelectropherograms for the stressed sample and the non-stressed sample;and

g. comparing the electropherograms from the stressed sample and thenonstressed sample, thereby determining the stability of the protein ofinterest;

wherein the stressed sample and the non-stressed sample are denatured,labeled and quenched in steps (b) through (d) prior to deglycoslation instep (e).

45. The method of embodiment 44, wherein stressing the sample comprisesthermally stressing the sample.

46. The method of embodiment 45, wherein thermally stressing the samplecomprises holding the sample at between about 30° C. and about 45° C.for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks or at least8 weeks.

47. The method of embodiment 44, wherein stressing the sample comprisesat least one freeze/thaw cycle.

48. The method of embodiment 44, wherein stressing the sample comprisesexposing the sample to storage conditions.

49. The methods of embodiment 48, wherein the storage conditionscomprise a temperature of about −80° C. to −30° C. for at least 1 week,at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months,at least 3 months, at least 6 months, at least 8 months, at least 12months, at least 18 months, at least 24 months or at least 30 months.

50. The methods of embodiment 48, wherein the storage conditionscomprise a temperature of about 2° C. to 8° C. for at least 1 week, atleast 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, atleast 3 months, at least 6 months, at least 8 months, at least 12 monthsor at least 18 months.

51. The method of embodiment 44, wherein stressing the sample comprisesmechanically agitating the sample.

52. The method of embodiment 44, wherein stressing the sample compriseslyophilizing and rehydrating the sample.

53. The method of embodiment 44, wherein stressing the sample comprisesexposing the sample to light, radiation, singlet oxygen species, freeradicals, high pH conditions or low pH conditions.

54. The method of any one of embodiments 44-53, wherein the protein ofinterest comprises at least one glycosylation site.

55. The method of any one of embodiments 44-53, wherein the protein isof interest is a glycosylated protein.

56. The method of embodiment 55, wherein the glycosylated proteincomprises at least one attached glycan.

57. The method of any one of embodiments 44-56, wherein the protein ofinterest comprises an antigen binding domain.

58. The method of embodiment 57, wherein the protein of interestcomprises an antibody, an antibody fragment or an scFv.

59. The method of any one of embodiments 44-58, wherein the protein ofinterest comprises an Fc domain.

60. The method of any one of embodiments 44-59, wherein the protein ofinterest comprises a receptor fusion protein.

61. The method of embodiment 60, wherein the receptor fusion protein isa receptor-Fc-fusion protein or a soluble TCR-Fc fusion protein.

62. The method of embodiment 60 or 61, wherein the receptor fusionprotein is a trap protein or a mini trap protein.

63. The method of any one of embodiments 44-62, wherein the protein ofinterest is a recombinant human protein.

64. The method of any one of embodiments 54-63, wherein theglycosylation site comprises an Asn-X-Ser/Thr consensus sequence.

65. The method of any one of embodiments 56-64, wherein the at least oneattached glycan is N-linked.

66. The method of embodiment 65, wherein the at least one attachedglycan is N-linked to an asparagine in the glycosylated protein.

67. The method of any one of embodiments 44-66, wherein theendoglycosidase catalyzes deglycosylation of N-linked glycans.

68. The method of embodiment any one of embodiments 44-67, wherein theendoglycosidase is selected from the group consisting ofPeptide-N-Glycosidase F (PNGase F), Endoglycosidase H (Endo H),Endoglycosidase S (Endo S), Endoglycosidase D, Endoglycosidase F1,Endoglycosidase F2 and Endoglycosidase F4.

69. The method of any one of embodiments 44-67, wherein theendoglycosidase is PNGase F.

70. The method of embodiment 69, wherein the PNGase F is Rapid PNGase F.

71. The method of embodiment 70, wherein the Rapid PNGase F isnon-reducing.

72. The method of any one of embodiments 44-71, wherein deglycosylatingthe stressed and non-stressed samples comprises heating the samples toabout 50° C. for 10 minutes.

73. The method of any one of embodiments 44-72, wherein deglycosylatingthe stressed and non-stressed samples comprises a reaction mixture foreach sample comprising between 0.2-1.5 mg labeled protein of interest,and between 1-5 μL Rapid PNGase F in a 10 μL reaction volume excludingthe volume of the Rapid PNGase F.

74. The method of embodiment 73, wherein the reaction mixture for eachof the stressed and non-stressed sample comprises 5 μL Rapid PNGase F.

75. The method of embodiment 73 or 74, wherein each of the stressed andnon-stressed sample comprise 0.2 mg labeled protein of interest.

76. The method of any one of embodiments 73-75, wherein the reactionmixture for each of the stressed and non-stressed sample comprises abuffer.

77. The method of any one of embodiments 44-63, wherein the at least oneglycan is an O-linked glycan.

78. The method of embodiment 77, wherein the endoglycosidase catalyzesdeglycosylation of O-linked glycans.

79. The method of embodiment 77 or 78, wherein the endoglycosidasecomprises Endo-α-N-acetylgalactosamindase (O-glycosidase).

80. The method of any one of embodiments 44-79, wherein labeling thestressed and non-stressed samples with the fluorescent label comprisesheating each sample to about 35° C. for 10-30 minutes.

81. The method of any one of embodiments 44-79, wherein labeling thestressed and non-stressed samples with the fluorescent label comprisesheating each sample to about 35° C. for about 15 minutes.

82. The method of any one of embodiments 44-81, wherein the stressed andnon-stressed samples are denatured using a reducing solution.

83. The method of embodiment 82, wherein the reducing solution comprisesdithiothreitol (DTT).

84. The method of any one of embodiments 44-81, wherein the stressed andnon-stressed samples are denatured using a non-reducing solution.

85. The method of embodiment 84, wherein the non-reducing solutioncomprises iodoacetamide (IAM).

86. The method of any one of embodiments 44-85, wherein denaturing thestressed and non-stressed samples comprises heating the samples tobetween 40° C. and 99° C. for between 1 minute and 5 hours.

87. The method of any one of embodiments 44-85, wherein denaturing thestressed and non-stressed samples comprises heating the samples tobetween 50° C. and 99° C. for between 1 to 60 minutes.

88. The method of any one of embodiments 44-87, wherein quenching theun-reacted fluorescent label comprises adding a stop solution.

89. The method of any one of embodiments 44-88, further comprisinganalyzing a reference standard in parallel to the stressed andnon-stressed samples.

90. The method of any one of embodiments 44-89, wherein comparing theelectropherograms for the stressed and non-stressed samples comprisescomparing peak number, height, position, area, or a combination thereof.

EXAMPLES Example 1: Reagents

Materials and Equipment

TABLE 1 Materials (equivalent items can also be used) Item VendorInformation and Handling Safe-Lock Eppendorf tubes, 1.5 mL VWR, cat. #21008-959 or 20901 548 96 well low skirted plates BioRad PN HSP-9621Millipore Ultrafree MC GV Durapore Cat. UFC30GVNB or Thermo ScientificPVDF 0.22 μM, or National Micro- (VWR Cat. 66064-450) centrifugalFilters, Non-sterile TX761 Swabs VWR PN TWTX761 VWR ® Heat-ResistantPolypropylene Film PN 89087-69 for Raised-Rim Plates Lint Free ClothWypall L40 PN Os701/7471 VWR Reagent Reservoir VWR 89094-674 Nalgene ®Bottle Top Filters, PES Thermo Scientific (VWR73521-002) Membrane,Sterile Protein Express LabChip, LabChip ® GXII, PN 760499 or 760528;LabChip ® GXII Touch ™ HT Store between 2-8° C. until use. Allow chip towarm for 30 min at room temperature before first time use. Once at roomtemperature, assign a 30 day expiration date to the chip.

TABLE 2 Chemicals (equivalent items can also be used) Chemical VendorInformation and Handling Water, purified by MilliQ — Protein ReferenceStandard Reference Standards be specific to an assay or experimentalprogram, or universal 0.2M Sodium Phosphate pH 8.0 VWR Cat. No. J6273310X Reducing Agent (0.5M Novex Life Technologies PN NP0009; Whendithiothreitol) received dispense stock as 1 mL aliquots and storebetween 2 and 8° C. Each vial should be used once and assigned a 6 monthexpiration Iodoacetamide (IAM) Sigma, A3221-10VL; Sigma, I1149 (MW184.96) (Store as a solid between 2 and 8° C.) Pico Protein Reagent KitPerkin Elmer PN 760498; The kit contains the following (vial cap colorsindicated and used throughout): Pico 5X Labeling Buffer (1 vial) (clear)Lyophilized Labeling Dye (4 vials) (blue) Sample Buffer (5 vials)(white) Protein Gel Matrix (2 vials) (red) Protein Ladder (1 vial)(yellow) Lower Marker (1 vial) (green) Wash Buffer (4 vials) (purple)Stop Buffer (1 vial) (orange) DMSO (Dimethyl sulfoxide) (1 vial) (brown)All reagents are stored between 2° C. and 8° C. except the LyophilizedLabeling Dye, which is stored at ≤−20° C. until reconstituted. The kitmust be held at room temperature for a minimum of 30 min prior to use.Sodium Phosphate Monobasic Sigma Aldrich Cat. No. 71504; Monohydrate (MW137.99) Store at room temperature Sodium Phosphate Dibasic Sigma AldrichCat. No. S2429; Heptahydrate (MW 268.07) Store at room temperatureLithium Dodecyl Sulfate (LDS) Sigma Aldrich Cat. No. L9781; (FW 272.33)(Store at room temperature) 70% Isopropanol (VWR 89108- for cleaning(Store at room temperature) 160) or Isopropanol

TABLE 3 Equipment Item Vendor Information and Handling Appropriatevolume pipettes and vendor VWR, Rainin or Gilson tips or equivalentMicrocentrifuge Eppendorf Model 5424 — or equivalent Plate CentrifugeModel 5804 equipped — for 96 well plates or equivalent EppendorfThermomixer for Eppendorf — tubes or Eppendorf Nexus Master Cycler withFlex lid for 96 well plates or equivalent Lab Chip GXII Perkin Elmer orLab Chip — GXII Touch HT Vortex or equivalent — Vacuum Aspiration Set upor equivalent Example set up - 1000 μL pipet tip attached to a firstpiece of plastic tubing, the tubing attached to a stoppered Erlenmeyerflask as a liquid reservoir, a second tube attaching the flask to avacuum source. The pipet tip is replaced after each cleaning step (e.g.,aspiration pass, sipper test)

TABLE 4 Reagent Solutions Reagent Solution Preparation Non-ReducingSolution: Prepare as a bulk solution and vortex to mix 272 μL 1M IAM1328 μL 100 mM Sodium Phosphate 1% LDS, pH 6 40 μL MilliQ water 1M IAM(Iodoacetamide) Add 303 μL MilliQ water to a 56 mg vial of IAM. Vortexuntil completely dissolved. Prepare fresh. Reducing Solution: Prepare asa bulk solution and vortex to mix 476 μL 10 x Reducing Agent 1162 μL 100mM Sodium Phosphate 1% LDS pH 9 42 μL MilliQ water Diluted StopSolution: Stop and Sample buffers from the Pico Protein 2.5 μL StopBuffer (orange cap) Reagent Kit 17.1 μL Sample Buffer (white cap) 85.4μL MilliQ water 5 μM Dye: Vortex 5 μM Dye Solution on high setting to 10μL of 100 μM Dye (frozen dissolve. aliquots stored at −20° C.) 190 μLMilliQ water 100 μM Dye Spin Lyophilized Labeling Dye (blue cap, PicoProtein Reagent Kit) at 15,000 rpm for 1 min. Add 240 μL of DMSO Vortexon high setting until completely dissolved. 200 mM Sodium Phosphate Add5.5 g of Sodium Phosphate Monobasic Monobasic Monohydrate Monohydrate to200 mL of MilliQ water. Mix until dissolved and filter through a 0.22 μmbottle top filter. 200 mM Sodium Phosphate Dibasic Add 10.7 g of SodiumPhosphate Dibasic Heptahydrate Heptahydrate to 200 mL of MilliQ water.Mix until dissolved and filter through a 0.22 μm bottle top filter. 10%LDS (lithium dodecyl sulfate) Dissolve 1 g of LDS in 8 mL of MilliQ andQS with MilliQ to a total volume of 10 mL. Filter through a 0.22 μmbottle top filter. 100 mM Sodium Phosphate 1% Mix solution using avortex. LDS pH 6: 8.18 mL 200 mM Sodium Phosphate Monobasic Monohydrate1.82 mL 200 mM Sodium Phosphate Dibasic Heptahydrate 2 mL 10% LDS 8 mLMilliQ water 100 mM Sodium Phosphate 1% Mix solution using a vortex. LDSpH 9: 10 mL 200 mM Sodium Phosphate Dibasic Heptahydrate 2 mL 10% LDS 8mL MilliQ water

TABLE 5 Summary of MCE methods MCE Method Protocol Description ResultsMethod A Example 2 No deglycosylation, No peak resolution forconventional sample heavily glycosylated preparation. protein; notstability indicating; Free dye interference at <20 kDa. Method B Example3 Deglycosylation prior to dye Good peak resolution; 3 labeling hoursdeglycosylation, PNGase F peak interference, free dye interference at<20k Da. Method C Example 4 Deglycosylation after dye Good peakresolution; no labeling PNGase F peak in profile; 10 mindeglycosylation, Resolution at 10-20 kDa, Stability indicating.

TABLE 6 Summary of Proteins used in the Examples MW Used in Used in(backbone Number of Protein Examples FIGs peptide) N-glycosylationDescription Protein 1 5, 6, 10 2-8, 49.4 8 Disulfide linked 14-15recombinant (Fab′)2-like trap protein Protein 2 7 9 48 8 Single ChainRecombinant (Fab′)2-like trap protein Protein 3 8 10-11 23 1 Isolated Fcfragment Protein 4 9 12-13 145 2 IgG4 mAb

Example 2: Protocol for Microchip Capillary Electrophoresis withoutDeglycosylation (Method A)

This protocol describes the preparation method for analysis of testproteins by non-reduced (NR) and reduced (R) Microchip CapillaryElectrophoresis (MCE) using the GXII instrument to estimate purity andimpurity levels. These methods are used for protein characterization ordetermining the level of fragmentation in a protein sample. Theseconventional methods are carried out without deglycosylation.

Procedure

See FIG. 1 for an information only flow path for this procedure.

(1) Denaturing. Dilute the protein reference standard or test articlewith water to about 0.2-2.0 mg/mL. In a 96-well plate, add the proteinsample and Non-Reducing (NR)/Reducing (R) Solution at a volume ratio of4:1 (volume can be varied). Seal the plate with polypropylene seal andheat the plate at a protein-specific denaturing temperature (typically50 to 99° C.) for an optimized time (typically 1 to 60 minutes).

(2) Labeling. Prepare the 5 μM dye as described in Table 4. Add the 5 μMdye to the denatured protein solution at a volume ratio of 1:1 (volumecan be varied). Heat the 96 well plate in a thermocycler at 35° C. for30 minutes. To quench the labeling reaction, add 105 μL of diluted stopsolution (prepared according to Table 4) and 5 μL of labeled protein toa new 96 well plate and mix well.

(3) Run on GX-II. Prepare the MCE instrument and microchip, and performthe measurements according to manufacturer's instructions.

Example 3: Protocol for Microchip Capillary Electrophoresis withDeglycosylation Before Protein Labeling (Method B)

This method applies to glycoproteins that need to be deglycosylatedbefore subjected to MCE measurements. Unless otherwise specified, allprotocols, chemicals, reagents and analysis are the same as described inExamples 1-2.

TABLE 7 Additional Reagents Reagent Vendor Information PNGase F NewEngland BioLabs NEB #P0704L GlycoBuffer 2 (10X) Buffer New EnglandBioLabs, #B3704 RapiGest SF Surfactant Waters, PN 186001861 Ammoniumbicarbonate (ABC) Sigma (Fluka), Cat#: 40867

Procedure

(1) Deglycosylation. Dilute a total 100 μg of the protein sample with0.1% RapiGest SF to 90 μL. Protein weight can be determined by UV basedmethods. Add 10 μL NEB PNGase F stock and make a 100 μL deglycosylationmixture, vortex and spin down. Incubate the mixture at 37° C. for 3hours on a heating block with shaking at 400 revolutions per minute(rpm).

(2) Denaturing. Proceed with denaturing of the above-mentioneddeglycosylated sample as described in Example 2.

(3) Labeling. Proceed with labeling the denatured sample as described inExample 2.

(4) Run on GX-II. Prepare the MCE instrument and microchip, and performthe measurements according to manufacturer's instructions.

Example 4: Protocol for Microchip Capillary Electrophoresis withDeglycosylation after Protein Labeling (Method C)

This method applies to glycoproteins that need to be deglycosylatedbefore subjected to MCE measurements. Unless otherwise specified, allprotocols, chemicals, reagents and analysis are the same as described inExamples 1-3.

A diagram of the protocol for deglycosylation after protein labeling canbe seen in FIG. 1.

TABLE 8 Additional Reagents Reagent Vendor Information Rapid ™ PNGase FNew England BioLabs (non-reducing format) NEB #P0710 5x Rapid ™ PNGase FBuffer New England BioLabs (non-reducing format) NEB #B0717S

Procedure

(1) Denaturing. Dilute and denature the sample as described in Example2.

(2) Labeling. Prepare the 5 μM dye as described in Table 4. Mix 5 μM dyeand above-mentioned denatured protein solution at a volume ratio of 1:1.For example, if the volume of the sample is 10 add 10 μL of 5 μM dye.Seal the 96 well plate with a polypropylene seal and heat in thethermocycler at 35° C. for 30 minutes. For quenching the labelingreaction, obtain an unused 96-well plate. Add 2.5 μL stop buffer (orangecap vial from the Pico Protein Reagent Kit, use the original solutionfrom the kit) to the wells of the empty plate according to the samplerun set up. Transfer 2.5 μL of labeled sample to the plate wellscontaining the stop solution. Pipet mix sample in each well and hold forat least 3 minutes.

(3) Deglycosylation. Add to each well 3 μL MilliQ water and 2 μL 5×Rapid™ PNGase Buffer (non-reducing format from NEB) to make a 10 μLreaction volume. Add to each well 1-4 μL Rapid™ PNGase (non-reducingformat from NEB). Seal the 96 well plate with a polypropylene seal andheat in the thermocycler at 50° C. for 10-30 minutes. Afterdeglycosylation, add to each well 17 μL Sample Buffer (white cap vialfrom the Pico Protein Reagent Kit) and 80 μL MilliQ water.

(4) Run on GX-II. Prepare the MCE instrument and microchip, and performthe measurements according to manufacturer's instructions.

Example 5: Comparing No Deglycosylation and Deglycosylation BeforeProtein Labeling Using a Heavily Glycosylated, Sialic Acid ContainingProtein

Protein 1 is a disulfide linked recombinant fusion protein which is 49kDa in size by peptide mass and has 8 predicted N-glycosylation sites(note, not all sites will be expected to be glycosylated).

One goal was to develop a microchip capillary electrophoresis-basedmethod to characterize and monitor low molecular weight (LMW) fragmentsof a heavily glycosylated, sialic acid containing protein (e.g.,Protein 1) for research stability studies and for quality control (QC)studies.

Characteristics of Protein 1 are shown in Table 9 below:

TABLE 9 Protein 1 Molecular Properties Molecular Weight without glycans49 kDa (Intact MS) Molecular Weight with glycans 64 kDa (SEC-MALS)Predicted N-linked glycosylation sites 8

Abbreviations: Intact MS, intact protein mass spectrometry; SEC-MALS,size exclusion chromatography multiple angle laser light scattering.

A comparison of a protocol without deglycosylation (Method A, Example 2)and a protocol with deglycosylation prior to labeling (Method B, Example3) is shown in FIG. 1. A comparison of the electropherograms of Protein1 produced by Method A and Method B is shown in FIG. 2 for non-reduced(NR) conditions, and in FIG. 4 for reduced (R), respectively. For NRconditions, as a result of Method A (without deglycosylation), there wasa broad peak in the electropherogram with no peak resolution (i.e., noseparation of Main, high molecular weight (HMW) and low molecular weight(LMW) peaks). In addition, the peak position appeared at a much highermolecular weight (MW) region (70-120 kDa) than what was expected atabout 64 kDa based on an orthogonal method. The MCE assay caninaccurately estimate size with a larger error when the protein isglycosylated (described by Engel et al. in Electrophoresis, 2015 August;36(15):1754-8). In addition, there was free dye peak interference at <20kDa that may mask any LMW peaks below 20 kDa.

In contrast, as result of Method B, where deglycosylation occurs priorto labeling (Example 3), there is peak resolution and baselineseparation between peaks (Main, HMW, LMW peaks). The Main peak appearedclose to the expected MW (about 49 kDa). The protocol is stabilityindicating and more accurate with molecular sizing. However, Method B'sprotocol also requires 3 hours of deglycosylation, which limits theoverall throughput of the assay. Moreover, the PNGase peak (about 36kDa) interferes with the LMW 1 and 2 peaks (impurity peaks fromfragments of Protein 1). Especially for thermally-stressed Protein 1samples, the LMW 1 peak increases and broadens, merging with PNGase peak(FIG. 3). Another nearby artifact is the free dye peak interference at<20 kDa. The combination of these artifacts can lead to inaccurateintegration when quantifying impurities and limits the assay's abilityto be stability indicating.

Similar observations were found for reduced conditions (FIG. 4):deglycosylation of glycoproteins is required for accurate sizing,separation and resolution of Main, LMW, and HMW peaks.

Example 6: Deglycosylation after Protein Labeling Using a HeavilyGlycosylated, Sialic Acid Containing Protein

A comparison of a protocols with deglycosylation before labeling (MethodB) and after labeling (Method C) is shown in FIG. 1.

To develop a protocol with deglycosylation after labeling, thedeglycosylation reaction conditions were optimized, as non-completeremoval of the glycosylated peak was initially observed with a 30-minutedeglycosylation reaction. Temperature, time, concentration and bufferconditions were varied to determine optimal deglycosylation of Protein1.

Optimization to remove the incompletely-deglycosylated peak in the NRelectropherogram of Protein 1 produced several improvements to theprotocol. These included using NEB Rapid™ PNGase F and performing thedeglycosylation at an elevated temperature, 50° C. for 10 minutes (usingconventional PNGase F needs 3 hours incubation at 37° C.), increasingthe endoglycosidase concentration, and adding Glycoprotein Buffer fromthe NEB PNGase kit.

Experiments showed increasing reaction time had no obvious improvementin deglycosylation. FIG. 5 shows electropherograms generated usingProtein 1, with 1 μL or 2 μL Rapid™ PNGase F (non-reducing format, NEBP0711), at 50° C., with reaction times varying from 10 to 30 minutes. Ascan be seen from FIG. 5, there was no obvious improvement (i.e.reduction of the incompletely deglycosylated peak) with reaction timesof more than 10 minutes.

Increasing Rapid™ PNGase F concentration improved deglycosylation. Usingthe protocol for deglycosylation after labeling, 1, 2, 3 or 4 μL ofRapid™ PNGase F were added to the deglycosylation reaction, and thereaction was allowed to proceed at 50° C. for 10 minutes. The resultsare shown in FIG. 6 and FIG. 7. As can be seen in the inset of FIG. 6,increasing the concentration of Rapid™ PNGase F decreases the Protein1's incomplete-deglycosylation shoulder peak.

Adding 1-4 μL of Rapid™ PNGase F provided robust results.Electropherograms generated using Reference Protein 1 deglycosylatedusing 1, 2, 3 or 4 μL of Rapid™ PNGase F are shown in FIG. 7. Area underthe indicated peaks was integrated using Empower, and the results areprovided in Table 10 below.

TABLE 10 Integration of Protein 1 peaks generated using differentamounts of Rapid ™ PNGase F Rapid  ™ PNGase LMW2-5 LMW 1 MP HMW 1 μL2.27 4.09 93.01 0.62 2 μL 1.94 4.23 93.24 0.60 3 μL 1.71 4.05 93.75 0.504 μL 1.78 4.10 93.38 0.75 % RSD 12.95 1.90 0.33 16.64

% RSD stands for percent relative standard deviation.

Integration results showed that although there was a decreasing shoulderpeak post main peak (incompletely deglycosylated Protein 1) that wasobserved with a higher Rapid™ PNGase F concentration, the totalpercentage of integration of the main peak (MP) had the highest value ataround 3 μL. From 1 μL to 4 μL of PNGase, the % RSD for the MP was0.33%, suggesting that using Rapid™ PNGase F concentrations of 1-4 μLper reaction provide robust results. Following this trend, using moreRapid™ PNGase is expected to provide similar results.

Method C, where deglycosylation occurs after labeling, enables this MCEassay to be both precise and stability indicating. Protein 1 wasstressed by holding the protein solution at 37° C. for 4 weeks (stressedReference Standard, or “SRS”, “37° C. 4 w” in Tables 11 and 12, comparedto “RS” or non-stressed Reference Standard), and assayed using thedeglycosylation following labeling protocol and MCE described here. Thisstressed Protein 1 was compared to non-stressed Protein 1 (time equal to0, or t0, i.e. no 37° C. hold). Electropherograms were generated forstressed and non-stressed Protein 1 (FIG. 8), and indicated peaks wereintegrated using Empower. Measurements were repeated for threereplicates (S1-S3), and the results are shown in Tables 11 and 12 below.

TABLE 11 Comparison of Stressed (SRS) and Non-Stressed (RS) Protein 1using a deglycosylation after labeling protocol LMW5 LMW4 LMW3 LMW2 LMW1MP HMW1 HMW2 Stress (%) (%) (%) (%) (%) (%) (%) (%) RS (t0) S1 0.11 0.200.75 0.81 4.22 93.36 0.54 — S2 0.07 0.17 0.70 0.80 4.22 93.57 0.48 — S30.12 0.17 0.73 0.81 4.21 93.38 0.57 — SRS S1 0.08 0.64 0.71 1.37 5.4689.25 2.34 0.15 (37° C., S2 0.10 0.63 0.71 1.39 5.52 89.25 2.19 0.20 4w) S3 0.16 0.68 0.68 1.35 5.45 89.63 1.87 0.18

TABLE 12 Percent Relative Standard Deviation (% RSD) of Stressed andNon-Stressed Protein 1 (N = 3) LMW (%) MP (%) HMW (%) RS (t0) Average6.03 93.36 0.54 % RSD 1.20 0.10 8.6 SRS (37° C., 4 w) Average 8.31 89.382.31 % RSD 0.55 0.25 9.98 Difference 2.28 −4.1 1.8

Three repeated measurements of RS and SRS showed multiple LMW and HMWpeaks were consistently identified between runs and integrated with lessthan 1% RSD for LMW and MP peak. All the changes were significant fromRS to SRS. A comparison of electropherograms from stressed, andnon-stressed Protein 1 prepared by Method C (FIG. 8) indicated that thismethod is precise and stability indicating.

When deglycosylation with PNGase F is performed before dye labeling(Method B), the PNGase F peak is visible in the electropherogram profileand interferes with the LMW 1 and LMW 2 peak. A long deglycosylationtime (3 hours) is used, and there is free dye interference (<20 kDa).

When deglycosylation with Rapid™ PNGase F is performed after labelingwith dye (Method C), no PNGase peak is visible in the electropherogram.There is fast and complete deglycosylation (e.g., in 10 minutes).Resolution of MP, HMW and LMW peaks is achieved along with minimal freedye interference down to about 10 kDa region (e.g. in FIG. 8, LMW 5 peakis 11 kDa and is baseline resolved from the free dye peak artifact).

In summary, MCE methods using deglycosylation after dye labeling, suchas Method C, have good resolution, are stability indicating, highthroughput, reproducible, and avoids assay artifacts from PNGase F Peakinterference and free dye interference. The methods also show goodprecision, linearity, and robustness. These assays can be used in aplate-based high throughput format which is suitable for quality controlpurposes.

Example 7: Comparison of MCE Results Generated with and withoutDeglycosylation Using Protein 2

Protein 2 is a single chain recombinant (Fab′)2-like protein comprisinga single chain fusion protein that includes a ligand binding domainlinked by a linker of sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ IDNO: 1). The protein has a predicted molecular weight of 48 kDa (peptidebackbone). Protein 2 has 8 N-glycosylation sites.

MCE electropherograms for Protein 2 were generated using a protocolwithout deglycosylation (Method A, Example 2), and with deglycosylationafter labeling (Method C, Example 4), under both non-reduced and reducedconditions. As can be seen in FIG. 9, without deglycosylation, there wasonly a broad peak without separation appearing at a MW region (90-140kDa) which was much larger than theoretical value (about 48 kDa). To thecontrary, deglycosylation produced a main peak near the expected MW (48kDa) and clearly resolved LMW peaks. Moreover, a PNGase peak (˜36 kDa)did not appear in the electropherogram.

Example 8: Comparison of MCE Results Generated with and withoutDeglycosylation Using Protein 3

Protein 3 is a recombinant human IgG 1 Fc subunit cleaved at a specificsite by a recombinant cysteine protease. The protein has a predictedmolecular weight of 23 kDa. The protein has one N-glycosylation site.

MCE electropherograms were generated using a protocol withoutdeglycosylation (Method A, Example 2), and with deglycosylation afterlabeling (Method C, Example 4), under non-reduced conditions. Theresults can be seen in FIG. 10. In the non-deglycosylation profile, twomain peaks (MPs) were found that represent non-glycosylated population(MP1, left) and glycosylated population (MP 2, right) in the originalsample. In the deglycosylated profile, only the non-glycosylated peakwas observed and several HMW peaks were resolved. The same comparisonwas also performed under reducing conditions (FIG. 11).

Example 9: Stability Assessment of a Monoclonal Antibody

Protein 4 is a human IgG4-based monoclonal antibody with a molecularweight of 145 kDa and 2 N-linked glycosylation sites.

MCE electropherograms were generated for Protein 4 samples preparedusing a protocol without deglycosylation (Method A, in FIGS. 12 and 13),and using a protocol with deglycosylation after labeling (Method C, inFIGS. 12 and 13). The protein was assayed using denaturation under bothnon-reducing conditions (FIG. 12) and reducing conditions (FIG. 13). Ascan be seen in FIG. 13, deglycosylation shifts the Glycosylated MainPeak (GMP) to a Deglycosylated Main Peak (DGMP) at a lower molecularweight as the result of the removal of glycans. In FIG. 13,deglycosylation reduces the size of the Heavy Chain (HC) peak, as can beseen by comparing the Deglycosylated Heavy Chain (DGHC) and GlycosylatedHeavy Chain (FGHC) peaks.

Example 10: Stability Assessment of Photo-Stressed Protein 1

Protein 1 was photo-stressed by exposing the protein solution under coolwhite (CW) fluorescent lamp light with 1.2 and 2.4 million lux hours(MLH) accumulative exposure (FIG. 14), or under integrated nearultraviolet (UVA) with an energy of 200 and 400 watt hours/square meter(FIG. 15). Samples were assayed using the deglycosylation followinglabeling protocol (Method C) and MCE as described in Examples 1 and 4.Stressed Protein 1 samples were compared to non-stressed Protein 1control (which was incubated under the same conditions but covered withaluminum foil). Electropherograms were generated for stressed andnon-stressed Protein 1, and indicated peaks were integrated usingEmpower. The results are shown in Table 13. Both CW and UVA exposure ledto slightly increases of LMW peaks and significant increases of HMWpeaks, which may due to photo-initiated formation of covalent-bondeddimers and multimers. The results show that this method is stabilityindicating and able to evaluate the fragmentation and covalent-bondedHMW formation of protein under stress conditions.

TABLE. 14 Comparison of photo stressed and non-stressed protein 1 usinga deglycosylation after labeling protocol (Method C). Photo stressconditions LMW (%) MP (%) HMW (%) Non-stressed 5.01 93.61 1.38 CW 1.2million lux hours 6.61 77.27 16.12 CW 2.4 million lux hours 7.54 69.9622.50 UVA 200 watt hours/m² 6.48 84.92 8.60

What is claimed is:
 1. A method of analyzing a sample comprising aprotein of interest, the method comprising: a. denaturing the sample; b.labeling the sample with a fluorescent label to produce a labeledsample; c. quenching un-reacted fluorescent label in the labeled sample;d. deglycosylating the labeled sample with an endoglycosidase; and e.performing electrophoresis on the labeled sample; wherein the sample isdenatured, labeled and quenched in steps (a) through (c) prior todeglycoslation in step (d).
 2. The method of claim 1, wherein theprotein of interest comprises at least one glycosylation site.
 3. Themethod of claim 1, wherein the protein is of interest is a glycosylatedprotein.
 4. The method of claim 1, wherein the protein of interestcomprises an antigen binding domain.
 5. The method of claim 4, whereinthe protein of interest comprises an antibody, an antibody fragment oran scFv.
 6. The method of claim 1, wherein the protein of interestcomprises an Fc domain.
 7. The method of claim 1, wherein the protein ofinterest comprises a receptor fusion protein.
 8. The method of claim 7,wherein the receptor fusion protein is a receptor-Fc-fusion protein or asoluble TCR-Fc fusion protein.
 9. The method of claim 1, wherein theprotein of interest is a recombinant human protein.
 10. The method ofclaim 1, wherein the protein of interest comprises at least one attachedglycan that is N-linked.
 11. The method of claim 1, wherein theendoglycosidase catalyzes deglycosylation of N-linked glycans.
 12. Themethod of claim 11, wherein the endoglycosidase is selected from thegroup consisting of Peptide-N-Glycosidase F (PNGase F), EndoglycosidaseH (Endo H), Endoglycosidase S (Endo S), Endoglycosidase D,Endoglycosidase F1, Endoglycosidase F2 and Endoglycosidase F4.
 13. Themethod of claim 11, wherein the endoglycosidase is PNGase F.
 14. Themethod of claim 13, wherein the PNGase F is Rapid PNGase F.
 15. Themethod of claim 14, wherein the Rapid PNGase F is non-reducing.
 16. Themethod of claim 13, wherein deglycosylating the sample comprises heatingthe sample to about 50° C. for 10 minutes.
 17. The method of claim 1,wherein deglycosylating the sample comprises a reaction mixturecomprising between 0.2-1.5 mg labeled protein of interest, and between1-5 μL Rapid PNGase F in a 10 μL reaction volume, excluding the volumeof the Rapid PNGase F.
 18. The method of claim 1, wherein the protein ofinterest comprises at least one glycan that is an O-linked glycan. 19.The method of claim 18, wherein the endoglycosidase catalyzesdeglycosylation of O-linked glycans.
 20. The method of claim 19, whereinthe endoglycosidase comprises Endo-α-N-acetylgalactosamindase(O-glycosidase).
 21. The method of claim 1, wherein labeling the samplewith the fluorescent label comprises heating the sample to about 35° C.for 10-30 minutes.
 22. The method of claim 1, wherein the sample isdenatured using a reducing solution.
 23. The method of claim 22, whereinthe reducing solution comprises dithiothreitol (DTT).
 24. The method ofclaim 1, wherein the sample is denatured using a non-reducing solution.25. The method of claim 24, wherein the non-reducing solution comprisesiodoacetamide (TAM).
 26. The methods of claim 1, wherein denaturing thesample comprises heating the sample to between 40° C. and 99° C. forbetween 1 minute and 60 minutes.
 27. The method of claim 1, whereinquenching the un-reacted fluorescent label comprises adding a stopsolution.
 28. The method of claim 1, further comprising analyzing areference standard in parallel to the sample.
 29. The method of claim 1,wherein the electrophoresis is selected from the group consisting of gelelectrophoresis, isoelectric focusing, capillary electrophoresis (CE) ormicrochip capillary electrophoresis (MCE).
 30. The method of claim 1,wherein method results in reduced free dye interference in the less than20 kDa range and a reduced or absent endoglycosidase peak in anelectropherogram when compared to an electropherogram generated using asample labeled after deglycosylation.
 31. A method of determiningstability of a protein of interest comprising: a. stressing a samplecomprising the protein of interest; b. denaturing the stressed sampleand a non-stressed sample comprising the protein of interest; c.labeling the stressed sample and the non-stressed sample with afluorescent label to produce a labeled stressed sample and a labelednon-stressed sample; d. quenching un-reacted fluorescent label in thelabeled stressed sample and the labeled non-stressed sample; e.deglycosylating the labeled stressed sample and the labeled non-stressedsample with an endoglycosidase; f. performing microchip capillaryelectrophoresis (MCE) on the labeled stressed sample and the labelednon-stressed sample to generate electropherograms for the stressedsample and the non-stressed sample; and g. comparing theelectropherograms from the stressed sample and the nonstressed sample,thereby determining the stability of the protein of interest; whereinthe stressed sample and the non-stressed sample are denatured, labeledand quenched in steps (b) through (d) prior to deglycoslation in step(e).